Genetically Modified T Cell Receptor Mice

ABSTRACT

The invention provides a genetically modified non-human animal that comprises in its genome unrearranged T cell receptor variable gene loci, as well as embryos, cells, and tissues comprising the same. Also provided are constructs for making said genetically modified non-human animal and methods of making the same. Various methods of using the genetically modified non-human animal are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 61/552,582, filed Oct. 28, 2011; U.S. ProvisionalApplication No. 61/621,198, filed Apr. 6, 2012; and U.S. ProvisionalApplication No. 61/700,908, filed Sep. 14, 2012, all of which areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

Present invention relates to a genetically modified non-human animal,e.g., a rodent (e.g., a mouse or a rat), that comprises in its genomehuman or humanized T Cell Receptor (TCR) variable gene loci (e.g., TCRαand TCRβ variable gene loci and/or TCRβ and TCRγ variable gene loci),and expresses human or humanized TCR polypeptides (e.g., TCRα and TCRβpolypeptides and/or TCRδ and TCRγ polypeptides) from the human orhumanized TCR variable gene loci. A non-human animal with human orhumanized TCR variable gene loci of the invention comprises unrearrangedhuman TCR variable region gene segments (e.g., V, D, and/or J segments)at endogenous non-human TCR gene loci. The invention also relates toembryos, tissues, and cells (e.g., T cells) that comprise human orhumanized TCR variable gene loci and express human or humanized TCRpolypeptides. Also provided are methods for making the geneticallymodified non-human animal comprising human or humanized TCR variablegene loci; and methods of using non-human animals, embryos, tissues, andcells that comprise human or humanized TCR variable gene loci andexpress human or humanized TCR polypeptides from those loci.

BACKGROUND OF THE INVENTION

In the adaptive immune response, foreign antigens are recognized byreceptor molecules on B lymphocytes (e.g., immunoglobulins) and Tlymphocytes (e.g., T cell receptors or TCRs). While pathogens in theblood and extracellular space are recognized by antibodies in the courseof humoral immune response, destruction of pathogens inside cells ismediated in the course of cellular immune response by T cells.

T cells recognize and attack antigens presented to them in the contextof a Major Histocompatibility Complex (MHC) on the cell surface. Theantigen recognition is mediated by TCRs expressed on the surface of theT cells. Two main classes of T cells serve this function: cytotoxic Tcells, which express a cell-surface protein CD8, and helper T cells,which express a cell-surface protein CD4. Cytotoxic T cells activatesignaling cascades that result in direct destruction of the cellpresenting the antigen (in the context of MHC I), while helper T cellsdifferentiate into several classes, and their activation (primed byrecognition of antigen presented in the context of MHC II) results inmacrophage-mediated pathogen destruction and stimulation of antibodyproduction by B cells.

Because of their antigen specificity, antibodies are presently widelystudied for their therapeutic potential against numerous humandisorders. To generate antibodies capable of neutralizing human targets,while simultaneously avoiding activation of immune responses againstsuch antibodies, scientists have concentrated their efforts on producinghuman or humanized immunoglobulins. One way of producing humanizedantibodies in vivo is by using VELOCIMMUNE® mouse, a humanized mousecomprising (1) unrearranged human immunoglobulin V, D, and J segmentrepertoire operably linked to each other and a mouse constant region atthe endogenous mouse immunoglobulin heavy chain locus and (2)unrearranged human Vκ and Jκ segment repertoire operably linked to eachother and a mouse constant κ region at the endogenous mouseimmunoglobulin κ light chain locus. As such, VELOCIMMUNE® mice provide arich source of highly diverse rearranged antibody variable domains foruse in engineering human antibodies.

Similar to an antibody, a T cell receptor comprises a variable region,encoded by unrearranged loci (α and β loci, or δ and γ loci) comprisingV(D)J variable region segments, and this variable region confers uponthe T cell its antigen binding specificity. Also similar to an antibody,the TCR specificity for its antigen can be utilized for development ofnovel therapeutics. Thus, there is a need in the art for non-humananimals (e.g., rodents, e.g., rats or mice) that comprise unrearrangedhuman T cell variable region gene segments capable of rearranging toform genes that encode human T cell receptor variable domains, includingdomains that are cognate with one another, and including domains thatspecifically bind an antigen of interest. There is also a need fornon-human animals that comprise T cell variable region loci thatcomprise conservative humanizations, including non-human animals thatcomprise unrearranged human gene segments that can rearrange to form Tcell receptor variable region genes that are linked to non-human(endogenous) T cell receptor constant gene sequences. There remains aneed for non-human animals that are capable of generating a diverserepertoire of human T cell receptor variable sequences. There is a needfor non-human animals that are capable of rearranging most or allfunctional T cell receptor variable region segments, in response to anantigen of interest, to form T cell receptor polypeptides that comprisefully human variable domains.

SUMMARY OF THE INVENTION

Non-human animals, e.g., rodents, comprising non-human cells thatexpress humanized molecules that function in the cellular immuneresponse are provided. Non-human animals that comprise unrearranged TCRvariable gene loci are also provided. In vivo and in vitro systems areprovided that comprise humanized rodent cells, wherein the rodent cellsexpress one or more humanized immune system molecules. Unrearrangedhumanized TCR rodent loci that encode humanized TCR proteins are alsoprovided.

In one aspect, provided herein is a genetically modified non-humananimal (e.g., a rodent, e.g., a mouse or a rat) that comprises in itsgenome (a) an unrearranged TCRα variable gene locus comprising at leastone human Vα segment and at least one human Jα segment, operably linkedto a non-human (e.g., a rodent, e.g., a mouse or a rat) TCRα constantgene sequence, and/or (b) an unrearranged TCRβ variable gene locuscomprising at least one human Vβ segment, at least one human Dβ segment,and at least one human Jβ segment, operably linked to a non-human (e.g.,a rodent, e.g., a mouse or a rat) TCRβ constant gene sequence.

In one embodiment, the unrearranged TCRα variable gene locus replacesendogenous non-human (e.g., rodent) TCRα variable gene locus at anendogenous TCRα variable gene locus. In one embodiment, the unrearrangedTCRβ variable gene locus replaces the endogenous non-human (e.g.,rodent) TCRβ variable gene locus at an endogenous TCRβ variable genelocus. In one embodiment, the endogenous non-human (e.g., rodent) Vα andJα segments are incapable of rearranging to form a rearranged Vα/Jαsequence. In one embodiment, the endogenous non-human (e.g., rodent) Vβ,Dβ, and Jβ segments are incapable of rearranging to form a rearrangedVβ/Dβ/Jβ sequence. In one embodiment, the non-human animal comprises adeletion such that the genome of the animal does not comprise afunctional Vα and functional Jα segment. In one embodiment, thenon-human animal comprises a deletion such that the genome of the animaldoes not comprise a functional endogenous Vβ, a functional endogenousDβ, and a functional endogenous Jβ segment. In one embodiment, theanimal comprises a deletion of all functional endogenous Vα and Jαsegments. In one embodiment, the rodent comprises a deletion of allfunctional endogenous Vβ, Dβ, and Jβ segments. In some embodiments, thehuman Vα and Jα segments rearrange to form a rearranged Vα/Jα sequence.In some embodiments, the human Vβ, Dβ, and Jβ segments rearrange to forma rearranged Vβ/Dβ/Jβ sequence. Thus, in various embodiments, thenon-human animal (e.g., rodent) expresses a T cell receptor comprising ahuman variable region and a non-human (e.g., rodent) constant region ona surface of a T cell.

In some aspects, T cells of the non-human animal undergo T celldevelopment in the thymus to produce CD4 and CD8 single positive Tcells. In some aspects, the non-human animal comprises a normal ratio ofsplenic CD3+ T cells to total splenocytes. In various embodiments, thenon-human animal generates a population of central and effector memory Tcells in the periphery.

In one embodiment, the unrearranged TCRα variable gene locus in thenon-human animal described herein comprises 61 human Jα segments and 8human Vα segments. In another embodiment, the unrearranged TCRα variablegene locus in the non-human animal comprises a complete repertoire ofhuman Jα segments and a complete repertoire of human Vα segments.

In one embodiment, the unrearranged TCRβ variable gene locus in thenon-human animal described herein comprises 14 human Jβ segments, 2human Dβ segments, and 14 human Vβ segments. In another embodiment, theunrearranged TCRβ variable gene locus in the non-human animal comprisesa complete repertoire of human Jβ segments, a complete repertoire ofhuman Dβ segments, and a complete repertoire of human Vβ segments.

In an additional embodiment, the non-human animal described herein(e.g., a rodent) further comprises nucleotide sequences of human TCRδvariable segments at a humanized TCRα locus. In one embodiment, thenon-human animal (e.g., rodent) further comprises at least one human Vδ,Dδ, and Jδ segments, e.g., a complete repertoire of human Vδ, Dδ, and Jδsegments at the humanized TCRα locus.

In one embodiment, the non-human animal retains an endogenous non-humanTCRα and/or TCRβ locus, wherein the locus is a non-functional locus.

In one embodiment, the non-human animal is a rodent. In one embodiment,the rodent is selected from a mouse and a rat. In one embodiment, therodent is a mouse.

In one aspect, the invention provides a genetically modified mousecomprising in its genome (a) an unrearranged TCRα variable gene locuscomprising a repertoire of human Jα segments and a repertoire of humanVα segments, operably linked to a non-human (e.g., mouse or rat) TCRαconstant gene sequence, and/or (b) an unrearranged TCRβ variable genelocus comprising a repertoire of human Jβ segments, a repertoire ofhuman Dβ segments, and a repertoire of human Vβ segments, operablylinked to a non-human (e.g., a mouse or rat) TCRβ constant genesequence. In one embodiment, the mouse comprises a complete repertoireof human Vα segments. In one embodiment, the mouse comprises a completerepertoire of human Vβ segments. In one embodiment, the mouse comprisesa complete repertoire of human Vα segments and human Jα segments. In oneembodiment, the mouse comprises a complete repertoire of human Vαsegments and human Vβ segments. In one embodiment, the mouse comprises acomplete repertoire of human Vα, human Jα, human Vβ, human Dβ, and humanJβ segments.

In one embodiment, the mouse comprises at least one endogenous mouse Vαand at least one endogenous mouse Jα segment, wherein the endogenoussegments are incapable of rearranging to form a rearranged Vα/Jαsequence, and also comprises at least one endogenous mouse Vβ, at leastone endogenous mouse Dβ, and at least one endogenous mouse Jβ segment,wherein the endogenous segments are incapable of rearranging to form arearranged Vβ/Dβ/Jβ sequence.

In one embodiment, the unrearranged TCRα variable gene locus thatcomprises human TCRα variable region segments replaces mouse TCRαvariable genes at the endogenous mouse TCRα variable locus, and theunrearranged TCRβ variable gene locus that comprises human TCRβ variableregion segments replaces mouse TCRβ variable genes at the endogenousmouse TCRβ variable locus.

In one embodiment, the human Vα and Jα segments rearrange to form arearranged human Vα/Jα sequence, and the human Vβ, Dβ, and Jβ segmentsrearrange to form a rearranged human Vβ/Dβ/Jβ sequence. In oneembodiment, the rearranged human Vα/Jα sequence is operably linked to amouse TCRα constant region sequence. In one embodiment, the rearrangedhuman Vβ/Dβ/Jβ sequence is operably linked to a mouse TCRβ constantregion sequence. Thus, in various embodiments, the mouse expresses a Tcell receptor on the surface of a T cell, wherein the T cell receptorcomprises a human variable region and a mouse constant region.

In one embodiment, the mouse further comprises a repertoire of humanTCRδ variable region segments (e.g., human Vδ, Jδ, and Dδ segments) at ahumanized TCRα locus. In one embodiment, the repertoire of human TCRδvariable region segments is a complete human TCRδ variable regionsegment repertoire. In one embodiment, the human TCRδ variable regionsegments are at the endogenous TCRα locus. In one embodiment, the humanTCRδ variable region segments replace endogenous mouse TCRδ variableregion segments.

In one embodiment, the genetically modified mouse expresses a T cellreceptor comprising a human variable region and a mouse constant regionon a surface of a T cell. In one aspect, the T cells of the mouseundergo thymic T cell development to produce CD4 and CD8 single positiveT cells. In one aspect, the mouse comprises a normal ratio of splenicCD3+ T cells to total splenocytes; in one aspect, the mouse generates apopulation of central and effector memory T cells to an antigen ofinterest.

Also provided are methods for making genetically modified non-humananimals (e.g., rodents, e.g., mice or rats) described herein.

In one aspect, a method for making a humanized rodent (e.g., a mouse orrat) is provided, comprising replacing rodent TCRα and TCRβ variableregion segments, but not rodent constant genes, with human unrearrangedTCRα and TCRβ variable region segments, at endogenous rodent TCR loci.In one embodiment, the method comprises replacing rodent TCRα variableregion segments (Vα and/or Jα) with human TCRα variable region segments(Vα and/or Jα), wherein the TCRα variable region segments are operablylinked to a non-human TCR constant region gene to form a humanized TCRαlocus; and replacing rodent TCRβ variable region segments (Vβ and/or Dβand/or Jβ) with human TCR variable region segments (Vβ and/or Dβ and/orJβ), wherein the TCR variable region segments are operably linked to anon-human TCR constant region gene to form a humanized TCRβ locus. Inone embodiment, the humanized rodent is a mouse and the germline of themouse comprises the human TCRα variable region segments operably linkedto an endogenous mouse TCRα constant sequence at an endogenous TCRαlocus; and the germline of the mouse comprises the human TCRβ variableregion segments operably linked to an endogenous mouse TCR constantsequence at an endogenous TCR locus.

In one embodiment, provided herein is a method for making a geneticallymodified non-human animal (e.g., rodent, e.g., mouse or rat) thatexpresses a T cell receptor comprising a human or humanized variableregion and a non-human (e.g., rodent) constant region on a surface of aT cell comprising: replacing in a first non-human animal an endogenousnon-human TCRα variable gene locus with an unrearranged humanized TCRαvariable gene locus comprising at least one human Vα segment and atleast one human Jα segment, wherein the humanized TCRα variable genelocus is operably linked to endogenous non-human TCRα constant region;replacing in a second non-human animal an endogenous non-human TCRvariable gene locus with an unrearranged humanized TCRβ variable genelocus comprising at least one human Vβ segment, at least one human Dβsegment, and at least one human Jβ segment, wherein the humanized TCRβvariable gene locus is operably linked to endogenous TCRβ constantregion; and breeding the first and the second non-human animal to obtaina non-human animal that expresses a T cell receptor comprising a humanor humanized variable region and a non-human constant region.

In one embodiment of the method, the endogenous non-human (e.g., rodent)Vα and Jα segments are incapable of rearranging to form a rearrangedVα/Jα sequence and the endogenous non-human (e.g., rodent) Vβ, Dβ, andJβsegments are incapable of rearranging to form a rearranged Vβ/Dβ/Jβsequence. In one embodiment of the method, the human Vα and Jα segmentsrearrange to form a rearranged Vα/Jα sequence and the human Vβ, Dβ, andJβ segments rearrange to form a rearranged Vβ/Dβ/Jβ sequence. In oneembodiment of the method, the unrearranged humanized TCRα variable genelocus comprises 61 human Jα segments and 8 human Vα segments, and theunrearranged humanized TCRβ variable gene locus comprises 14 human Vβsegments, 2 human Dβ segments, and 14 human Jβ segments. In anotherembodiment of the method, the unrearranged humanized TCRα variable genelocus comprises a complete repertoire of human Jα segments and acomplete repertoire of human Vα segments, and the unrearranged humanizedTCR variable gene locus comprises a complete repertoire of human Vβsegments, a complete repertoire of human Dβ segments, and a completerepertoire of human Jβ segments.

In one aspect of the method, the T cells of the non-human animal (e.g.,rodent) undergo thymic T cell development to produce CD4 and CD8 singlepositive T cells. In one aspect, the non-human animal (e.g., the rodent)comprises a normal ratio of splenic CD3+ T cells to total splenocytes.In one aspect, the non-human animal (e.g., rodent) generates apopulation of central and effector memory T cells to an antigen ofinterest.

In some embodiments, the replacement of the endogenous non-human TCRαvariable gene locus described herein is made in a single ES cell, andthe single ES cell is introduced into a non-human (e.g., a rodent, e.g.,a mouse or rat) embryo to make a genetically modified non-human animal(i.e., the first non-human animal, e.g., the first rodent); and thereplacement of the endogenous non-human TCRβ variable gene locusdescribed herein is made in a single ES cell, and the single ES cell isintroduced into a non-human (e.g., a rodent, e.g., a mouse or rat)embryo to make a genetically modified non-human animal (i.e., the secondnon-human animal, e.g., the second rodent). In one embodiment, the firstrodent and the second rodent are bred to form a progeny, wherein theprogeny comprises in its germline a humanized TCRα variable locus and ahumanized TCR variable locus.

In one embodiment of the method, the non-human animal is a rodent, e.g.,a mouse. Thus, the present invention also provides a method for making agenetically modified mouse.

Also provided herein are cells, e.g., isolated T cells (e.g., cytotoxicT cells, helper T cells, memory T cells, etc.), derived from thenon-human animals (e.g., rodents, e.g., mice or rats) described herein.Tissues and embryos derived from the non-human animals described hereinare also provided.

In one aspect, a method for making a human TCR variable domain isprovided, comprising genetically modifying a rodent as described hereinto comprise a humanized TCRα locus and/or a humanized TCRβ locus,maintaining the rodent under conditions sufficient to form a T cell,wherein the T cell expresses a human TCRα and/or a human TCRβ variabledomain.

In one aspect, a method for making a nucleic acid sequence encoding ahuman TCR variable domain that binds an epitope of interest is provided,comprising exposing a non-human animal as described herein to an epitopeof interest, maintaining the non-human animal under conditionssufficient for the animal to present the epitope of interest to ahumanized TCR of the animal, and identifying a nucleic acid of theanimal that encodes a human TCR variable domain polypeptide that bindsthe epitope of interest.

In one aspect, use of a non-human animal as described herein is providedfor making a humanized TCR receptor. In one aspect, use of a non-humananimal as described herein is provided for making a human TCR variabledomain. In one aspect, use of a non-human animal as described herein isprovided for making a nucleic acid sequence encoding a human TCRvariable domain.

In one aspect, use of nucleic acid sequence encoding a human TCRvariable domain or fragment thereof to make an antigen-binding proteinis provided. In one embodiment, the antigen-binding protein comprises aTCR variable domain comprising a human TCRα and/or human TCRβ variabledomain that binds an antigen of interest.

In one aspect, use of a non-human as described herein is provided formaking a non-human cell that expresses on its surface a humanized T cellreceptor.

In one aspect, a humanized T cell receptor from a non-human animal asdescribed herein is provided.

In one aspect, a nucleic acid sequence encoding a human TCR variabledomain or fragment thereof, made in a non-human animal as describedherein, is provided.

Any of the embodiments and aspects described herein can be used inconjunction with one another, unless otherwise indicated or apparentfrom the context. Other embodiments will become apparent to thoseskilled in the art from a review of the ensuing detailed description.The following detailed description includes exemplary representations ofvarious embodiments of the invention, which are not restrictive of theinvention as claimed. The accompanying figures constitute a part of thisspecification and, together with the description, serve only toillustrate embodiments and not to limit the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts interaction in a mouse between a TCR molecule and an MHCmolecule: the left panel shows a mouse T cell (top) from a humanized TCRmouse comprising a T cell receptor with human variable TCR domains andmouse constant TCR domains, which recognizes an antigen (grey ball)presented through an MHC class I by an antigen presenting cell (bottom);the right panel shows the same for an MHC class II. The MHC I and MHC IIcomplexes are shown together with their respective co-receptors, CD8 andCD4. Mouse regions are in black and human regions are in white.

FIG. 2 depicts (not to scale) the general organization of a mouse (toppanel, first locus) and human (top panel, second locus) TCRα locus. Thebottom panel illustrates a strategy for replacing TCRα variable regionsegments in a mouse (closed symbols) with human TCRα variable regionsegments (open symbols) at the endogenous mouse locus on chromosome 14;a humanized TCRα locus having human Vα and Jα segments is shown with amouse constant region and a mouse enhancer; in the embodiment shown, theTCRβ locus is deleted in the course of humanization.

FIG. 3 depicts (not to scale) a progressive strategy for humanization ofthe mouse TCRα locus, wherein TCRα variable region gene segments aresequentially added upstream of an initial humanization of a deletedmouse locus (MAID1540). Mouse sequence is indicated by closed symbols;human sequence is indicated by open symbols. MAID refers to modifiedallele ID number. TRAV=TCR Vα segment, TRAJ=TCR Jα segment (hTRAJ=humanTRAJ), TRAC=TCR Cα domain, TCRD=TCRδ.

FIG. 4 is a detailed depiction (not to scale) of progressivehumanization strategy at the TCRα locus. FIG. 4A depicts deletion of themouse TCRα V and J segments; FIG. 4B depicts strategy for insertion of2V and 61J human segments into the deleted mouse TCRα locus; FIG. 4Cdepicts strategy for insertion of additional human V segments, resultingin a total of 8V and 61J human segments; FIG. 4D depicts strategy forinsertion of additional human V segments, resulting in a total of 23Vand 61J human segments; FIG. 4E depicts strategy for insertion ofadditional human V segments resulting in 35V and 61J human segments;FIG. 4F depicts strategy for insertion of additional human segmentsresulting in 48V and 61J human segments; and FIG. 4G depicts strategyfor insertion of additional human segments resulting in 54V and 61Jhuman segments. MAID refers to modified allele ID number.

FIG. 5 depicts (not to scale) one embodiment of mouse TCRα locushumanization strategy, in which human TCR 8 sequences (TORδ Vs, TCRδ Ds,TCRδ Js, TCRδ enh (enhancer), and TCRδ constant (C)) are also placed atthe humanized TCRα locus. Mouse sequence is indicated by closed symbols;human sequence is indicated by open symbols. LTVEC refers to a largetargeting vector; hTRD=human TCRδ.

FIG. 6 depicts (not to scale) the general organization of a mouse (toppanel, first locus; on mouse chromosome 6) and human (top panel, secondlocus; on human chromosome 7) TCRβ loci. The bottom panel illustrates astrategy for replacing TCRβ variable region segments in the mouse(closed symbols) with human TCRβ variable region segments (open symbols)at the endogenous mouse locus on mouse chromosome 6. The humanized TCRβlocus having human Vβ, Dβ, and Jβ segments is shown with mouse constantregions and a mouse enhancer; in the embodiment shown, the humanizedlocus retains mouse trypsinogen genes (solid rectangles); and in theparticular embodiment shown, a single mouse V segment is retainedupstream of the 5′ mouse trypsinogen genes.

FIG. 7 depicts (not to scale) a progressive strategy for humanization ofthe mouse TCRβ locus, wherein TCRβ variable region gene segments aresequentially added to a deleted mouse TCRβ variable locus. Mousesequence is indicated by closed symbols; human sequence is indicated byopen symbols. MAID refers to modified allele ID number. TRBV orTCRBV=TCRβ V segment.

FIG. 8 is a detailed depiction of progressive humanization strategy atthe TCRβ locus. FIG. 8A depicts the strategy for deletion of the mouseTCRβ V segments; FIG. 8B depicts the strategy for insertion of 14Vsegments into the deleted TCRβ locus; FIG. 8C depicts the strategy forinsertion of 2D and 14J segments into TCRβ locus (i), followed bydeletion of the loxP site (ii), resulting in 14V, 2D and 14J humansegments; FIG. 8D depicts the strategy for inserting additional human Vsegments resulting in 40V, 2D and 14J human segments; and FIG. 8Edepicts the strategy for insertion of additional human V segmentsresulting in the 66V, 2D and 14J human segments; FIG. 8F depictsreplacement of the mouse V segment downstream of a mouse enhancer,resulting in 67V, 2D and 14J human segments. In this particularembodiment, one mouse V segment is retained 5′ of the mouse trypsinogengenes.

FIG. 9 depicts representative FACS analysis histograms for percentspleen cells (where Y axis is number of cells, X axis is meanfluorescence intensity, and the gate shows frequency of CD3+ T cellswithin the single lymphocyte population) stained with anti-CD3 antibodyin a wild type (WT) mouse; a mouse homozygous for a deleted TCRα locus(first top panel; MAID 1540 of FIG. 3); a mouse homozygous for a deletedTCRα locus and comprising 8 human Vα and 61 human Jα segments (secondtop panel; MAID 1767 of FIG. 3 or a humanized TCRα mouse); a mousehomozygous for a deleted TCRβ locus with the exception of one upstreamand one downstream mouse Vβ segments (first bottom panel; MAID 1545 ofFIG. 7); a mouse homozygous for a deleted TCRβ locus with one upstreamand one downstream mouse Vβ segments and comprising 14 human Vβ, 2 humanDβ, and 14 human Jβ segments (second bottom panel; MAID 1716 of FIG. 7or a humanized TCRβ mouse); and a mouse homozygous for both TCRα andTCRβ loci deletions (with the exception of said two mouse Vβ segments)and comprising 8 human Vα and 61 human Jα segments at the endogenousTCRα locus as well as 14 human Vβ, 2 human Dβ, and 14 human Jβ segmentsat the endogenous TCRβ loci (MAID 1767/1716 or a humanized TCRα/βmouse).

FIG. 10 is a representative FACS contour plot of mouse thymus cells froma WT, homozygous humanized TCRα (1767 HO; hTCRα); homozygous humanizedTCRβ (1716 HO; hTCRβ); and homozygous humanized TCRα/β mouse (1716 HO1767 HO; hTCRα/β) stained with anti-CD4 (Y axis) and anti-CD8 (X axis)antibodies (top panel), and anti-CD44 (Y axis) and anti-CD25 (X axis)antibodies (bottom panel). The FAGS plot in the top panel allows todistinguish double negative (DN), double positive (DP), CD4 singlepositive (CD4 SP), and CD8 single positive (SP CD8) T cells. The FACSplot in the bottom panel allows to distinguish various stages of doublenegative T cells during T cell development (DN1, DN2, DN3, and DN4).1716 and 1767 refer to MAID numbers as identified in FIGS. 3 and 7.

FIG. 11 demonstrates either frequency (top panel) or absolute number(bottom panel) of DN, DP, CD4 SP, and CD SP T cells in the thymus ofeither WT, hTCRα (1767 HO); hTCRβ (1716 HO); or hTCRα/β (1716 HO 1767HO) mice (n=4).

FIG. 12 is a representative FACS analysis of spleen cells of a WT, hTCRα(1767 HO); hTCRβ (1716 HO); or hTCRα/β (1716 HO 1767 HO) mouse: leftpanel represents analysis of singlet cells based anti-CD19 antibody (Yaxis; stain for B lymphocytes) or anti-CD3 antibody (X axis; stain for Tlymphocytes) staining; middle panel represents analysis of CD3+ cellsbased on anti-CD4 (Y axis) or anti-CD8 (X axis) antibody staining; andright panel represents analysis of either CD4+ or CD8+ cells based onanti-CD44 (Y axis) or anti-CD62L (X axis) antibody staining, the stainsallow to distinguish various types of T cells in the periphery (naïve Tcells vs. central memory T cells (Tcm) vs. effector or effector memory Tcells (Teff/Tem)).

FIG. 13 demonstrates the number of CD4+ (left panel) or CD8+ (rightpanel) T cells per spleen (Y axes) of WT, hTCRα (1767 HO); hTCRβ (1716HO); or hTCRα/β (1716 HO 1767 HO) mice (n=4).

FIG. 14 demonstrates the number of T naïve, Tcm, and Teff/em cells perspleen (Y axes) of CD4+ (top panel) or CD8+ (bottom panel) T cells ofWT, hTCRα (1767 HO); hTCRβ (1716 HO); or hTCRα/β (1716 HO 1767 HO) mice(n=4).

FIG. 15 are tables summarizing expression (determined by FACS analysisusing variable segment-specific antibodies) of various human TCRβ Vsegments in the splenic CD8+ T cells (FIG. 15A) or CD4+ T cells (FIG.15B) of WT, hTCRβ (1716 HO) or hTCRα/β (1716 HO 1767 HO) mice. Datapresented as Mean±SD (n=4 mice per group)

FIG. 16 depicts mRNA expression (Y axes) of various human TCRβ Vsegments present in WT, hTCRα (1767 HO); hTCRβ (1716 HO); or hTCRα/β(1716 HO 1767 HO) mice in thymic or splenic T cells; FIG. 16A representsanalysis of mRNA expression of human TCRβ variable segment (hTRBV) 18,19, 20, and 24; and FIG. 16B represents analysis of mRNA expression ofhTRBV 25, 27, 28, and 29.

FIG. 17 depicts representative FACS histograms of spleen cells (where Yaxis is number of cells, X axis is mean fluorescence intensity, and thegate shows frequency of CD3+ T cells within the single lymphocytepopulation) stained with anti-CD3 antibody in a WT mouse, a mousehomozygous for a deleted TCRα locus (TCRA ΔV), a mouse homozygous fordeleted TCRα locus with 2 human V segments and 61 human J segments (TCRA2 hV; MAID 1626 of FIG. 3), a mouse homozygous for deleted TCRα locuswith 8 human V segments and 61 human J segments (TCRA 8 hV; MAID 1767 ofFIG. 3), and a mouse homozygous for deleted TCRα locus with 23 human Vsegments and 61 human J segments (TCRA 23 hV; MAID 1979 of FIG. 3).

FIG. 18, at left top panel, is a representative FACS analysis of CD3+ Tcells of the thymus obtained from either a WT or homozygous hTCRα mousewith 23 human V segments and 61 human J segments (1979 HO) stained witheither anti-CD4 (Y axis) or anti-CD8 (X axis) antibody; at left bottompanel, is a FACS analysis of DN T cells from either a WT or 1979 mousestained with either anti-CD44 (Y axis) or anti-CD25 (X axis); at rightpanel are graphs of percent of thymocytes (Y axis) that are DN, DP, CD4SP, or CD8 SP in either WT or 1979 HO mice (n=4).

FIG. 19, at left panel, is a representative FACS analysis of spleniclymphocytes from a WT or 1979 HO mouse stained either with anti-CD19 oranti-CD3 antibodies; at right panel, are graphs of percent splenocytes(Y axis) obtained from WT and 1979 HO mice (n=4) that are CD3+.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The present invention provides genetically modified non-human animals,e.g., rodents, e.g., mice or rats, that express humanized T cellreceptors. The present invention also relates to genetically modifiednon-human animals that comprise in their germline unrearranged T cellreceptor variable gene loci. Also provided are embryos, cells, andtissues comprising the same; methods of making the same; as well asmethods of using the same. Unless defined otherwise, all terms andphrases used herein include the meanings that the terms and phrases haveattained in the art, unless the contrary is clearly indicated or clearlyapparent from the context in which the term or phrase is used.

The term “conservative,” when used to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). Conservative amino acidsubstitutions may be achieved by modifying a nucleotide sequence so asto introduce a nucleotide change that will encode the conservativesubstitution. In general, a conservative amino acid substitution willnot substantially change the functional properties of interest of aprotein, for example, the ability of a T cell to recognize a peptidepresented by an MHC molecule. Examples of groups of amino acids thathave side chains with similar chemical properties include aliphatic sidechains such as glycine, alanine, valine, leucine, and isoleucine;aliphatic-hydroxyl side chains such as serine and threonine;amide-containing side chains such as asparagine and glutamine; aromaticside chains such as phenylalanine, tyrosine, and tryptophan; basic sidechains such as lysine, arginine, and histidine; acidic side chains suchas aspartic acid and glutamic acid; and, sulfur-containing side chainssuch as cysteine and methionine. Conservative amino acids substitutiongroups include, for example, valine/leucine/isoleucine,phenylalanine/tyrosine, lysine/arginine, alanine/valine,glutamate/aspartate, and asparagine/glutamine. In some embodiments, aconservative amino acid substitution can be a substitution of any nativeresidue in a protein with alanine, as used in, for example, alaninescanning mutagenesis. In some embodiments, a conservative substitutionis made that has a positive value in the PAM250 log-likelihood matrixdisclosed in Gonnet et al. ((1992) Exhaustive Matching of the EntireProtein Sequence Database, Science 256:1443-45), hereby incorporated byreference. In some embodiments, the substitution is a moderatelyconservative substitution wherein the substitution has a nonnegativevalue in the PAM250 log-likelihood matrix.

Thus, encompassed by the invention is a genetically modified non-humananimal expressing humanized TCR α and β polypeptides (and/or humanizedTCRδ and TCRγ polypeptides) comprising conservative amino acidsubstitutions in the amino acid sequence described herein.

One skilled in the art would understand that in addition to the nucleicacid residues encoding humanized TCR α and β polypeptides describedherein, due to the degeneracy of the genetic code, other nucleic acidsmay encode the polypeptides of the invention. Therefore, in addition toa genetically modified non-human animal that comprises in its genomenucleotide sequences encoding humanized TCR polypeptides describedherein, a non-human animal that comprises in its genome nucleotidesequences that differ from those described herein due to the degeneracyof the genetic code are also provided.

The term “identity” when used in connection with sequence includesidentity as determined by a number of different algorithms known in theart that can be used to measure nucleotide and/or amino acid sequenceidentity. In some embodiments described herein, identities aredetermined using a ClustalW v. 1.83 (slow) alignment employing an opengap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnetsimilarity matrix (MacVector™ 10.0.2, MacVector Inc., 2008). The lengthof the sequences compared with respect to identity of sequences willdepend upon the particular sequences. In various embodiments, identityis determined by comparing the sequence of a mature protein from itsN-terminal to its C-terminal. In various embodiments, when comparing achimeric human/non-human sequence to a human sequence, the human portionof the chimeric human/non-human sequence (but not the non-human portion)is used in making a comparison for the purpose of ascertaining a levelof identity between a human sequence and a human portion of a chimerichuman/non-human sequence (e.g., comparing a human ectodomain of achimeric human/mouse protein to a human ectodomain of a human protein).

The terms “homology” or “homologous” in reference to sequences, e.g.,nucleotide or amino acid sequences, means two sequences which, uponoptimal alignment and comparison, are identical in at least about 75% ofnucleotides or amino acids, at least about 80% of nucleotides or aminoacids, at least about 90-95% nucleotides or amino acids, e.g., greaterthan 97% nucleotides or amino acids. One skilled in the art wouldunderstand that, for optimal gene targeting, the targeting constructshould contain arms homologous to endogenous DNA sequences (i.e.,“homology arms”); thus, homologous recombination can occur between thetargeting construct and the targeted endogenous sequence.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. As such, a nucleic acid sequenceencoding a protein may be operably linked to regulatory sequences (e.g.,promoter, enhancer, silencer sequence, etc.) so as to retain propertranscriptional regulation. In addition, various portions of thehumanized protein of the invention may be operably linked to retainproper folding, processing, targeting, expression, and other functionalproperties of the protein in the cell. Unless stated otherwise, variousdomains of the humanized protein of the invention are operably linked toeach other.

The term “replacement” in reference to gene replacement refers toplacing exogenous genetic material at an endogenous genetic locus,thereby replacing all or a portion of the endogenous gene with anorthologous or homologous nucleic acid sequence. In one instance, anendogenous non-human gene or fragment thereof is replaced with acorresponding human gene or fragment thereof. A corresponding human geneor fragment thereof is a human gene or fragment that is an ortholog of,a homolog of, or is substantially identical or the same in structureand/or function, as the endogenous non-human gene or fragment thereofthat is replaced. As demonstrated in the Examples below, nucleotidesequences of endogenous non-human TCR α and β variable gene loci werereplaced by nucleotide sequences corresponding to human TCR α and βvariable gene loci.

“Functional” as used herein, e.g., in reference to a functional protein,refers to a protein that retains at least one biological activitynormally associated with the native protein. For example, in someembodiments of the invention, a replacement at an endogenous locus(e.g., replacement at endogenous non-human TCRα, TCRβ, TCRδ and/or TCRγvariable gene loci) results in a locus that fails to express afunctional endogenous protein.

TCR locus or TCR gene locus (e.g., TCRα locus or TCRβ locus), as usedherein, refer to the genomic DNA comprising the TCR coding region,including the entire TCR coding region, including unrearranged V(D)Jsequences, enhancer, sequence, constant sequence(s), and any upstream ordownstream (UTR, regulatory regions, etc.), or intervening DNA sequence(introns, etc.). TCR variable locus or TCR variable gene locus (e.g.,TCRα variable gene locus or TCRβ variable gene locus), refers to genomicDNA comprising the region that includes TCR variable region segments(V(D)J region) but excludes TCR constant sequences and, in variousembodiments, enhancer sequences. Other sequences may be included in theTCR variable gene locus for the purposes of genetic manipulation (e.g.,selection cassettes, restriction sites, etc.), and these are encompassedherein.

Genetically Modified TCR Animals

In various embodiments, the invention generally provides geneticallymodified non-human animals wherein the non-human animals comprise in thegenome unrearranged humanized TCR variable gene loci.

T cells bind epitopes on small antigenic determinants on the surface ofantigen-presenting cells that are associated with a majorhistocompatibility complex (MHC; in mice) or human leukocyte antigen(HLA; in humans) complex. T cells bind these epitopes through a T cellreceptor (TCR) complex on the surface of the T cell. T cell receptorsare heterodimeric structures composed of two types of chains: an α(alpha) and β (beta) chain, or a γ (gamma) and δ (delta) chain. The αchain is encoded by the nucleic acid sequence located within the α locus(on human or mouse chromosome 14), which also encompasses the entire δlocus, and the β chain is encoded by the nucleic acid sequence locatedwithin the β locus (on mouse chromosome 6 or human chromosome 7). Themajority of T cells have an αβ TCR; while a minority of T cells bear aγδ TCR. Interactions of TCRs with MHC class I (presenting to CD8+ Tcells) and MHC class II (presenting to CD4+ T cells) molecules are shownin FIG. 1 (closed symbols represent non-human sequences; open symbolsrepresent human sequences, showing one particular embodiment of the TCRprotein of the present invention).

T cell receptor α and β polypeptides (and similarly γ and δpolypeptides) are linked to each other via a disulfide bond. Each of thetwo polypeptides that make up the TCR contains an extracellular domaincomprising constant and variable regions, a transmembrane domain, and acytoplasmic tail (the transmembrane domain and the cytoplasmic tail alsobeing a part of the constant region). The variable region of the TCRdetermines its antigen specificity, and similar to immunoglobulins,comprises 3 complementary determining regions (CDRs). Also similar toimmunoglobulin genes, T cell receptor variable gene loci (e.g., TCRα andTCRβ loci) contain a number of unrearranged V(D)J segments (variable(V), joining (J), and in TCRβ and δ, diversity (D) segments). During Tcell development in the thymus, TCRα variable gene locus undergoesrearrangement, such that the resultant TCR a chain is encoded by aspecific combination of VJ segments (Vα/Jα sequence); and TCRβ variablegene locus undergoes rearrangement, such that the resultant TCR β chainis encoded by a specific combination of VDJ segments (Vβ/Dβ/Jβsequence).

Interactions with thymic stroma trigger thymocytes to undergo severaldevelopmental stages, characterized by expression of various cellsurface markers. A summary of characteristic cell surface markers atvarious developmental stages in the thymus is presented in Table 1.Rearrangement at the TCRβ variable gene locus begins at the DN2 stageand ends during the DN4 stage, while rearrangement of the TCRα variablegene locus occurs at the DP stage. After the completion of TCRβ locusrearrangement, the cells express TCRβ chain at the cell surface togetherwith the surrogate α chain, pTα. See, Janeway's Immunobiology, Chapter7, supra.

TABLE 1 Developmental Stages of T cells in the Thymus DevelopmentalStage DN1 DN2 DN3 DN4 DP SP Marker(s) CD44+/ CD44+/ CD44^(low)/ CD44−/CD4+/ CD4+ or CD25− CD25+ CD25+ CD25− CD8+ CD8+

Naive CD4+ and CD8+ T cells exit the thymus and enter the peripherallymphoid organs (e.g., spleen) where they are exposed to antigens andare activated to clonally expand and differentiate into a number ofeffector T cells (Teff), e.g., cytotoxic T cells, T_(REG) cells, T_(H)17cells, T_(H)1 cells, T_(H)2 cells, etc. Subsequent to infection, anumber of T cells persist as memory T cells, and are classified aseither central memory T cells (Tcm) or effector memory T cells (Tern).Sallusto et al. (1999) Two subsets of memory T lymphocytes with distincthoming potentials and effector functions, Nature 401:708-12 andCommentary by Mackay (1999) Dual personality of memory T cells, Nature401:659-60. Sallusto and colleagues proposed that, after initialinfection, Tem cells represent a readily available pool ofantigen-primed memory T cells in the peripheral tissues with effectorfunctions, while Tcm cells represent antigen-primed memory T cells inthe peripheral lymphoid organs that upon secondary challenge can becomenew effector T cells. While all memory T cells express CD45RO isoform ofCD45 (naïve T cells express CD45RA isoform), Tcm are characterized byexpression of L-selectin (also known as CD62L) and CCR7+, which areimportant for binding to and signaling in the peripheral lymphoid organsand lymph nodes. Id. Thus, all T cells found in the peripheral lymphoidorgans (e.g., naïve T cells, Tcm cells, etc.) express CD62L. In additionto CD45RO, all memory T cells are known to express a number of differentcell surface markers, e.g., CD44. For summary of various cell surfacemarkers on T cells, see Janeway's Immunobiology, Chapter 10, supra.

While TCR variable domain functions primarily in antigen recognition,the extracellular portion of the constant domain, as well astransmembrane, and cytoplasmic domains of the TCR also serve importantfunctions. A complete TCR receptor complex requires more than the α andβ or γ and δ polypeptides; additional molecules required include CD3γ,CD3δ, and CD3ε, as well as the chain ζ homodimer (ζζ). At the completionof TCRβ rearrangement, when the cells express TCRβ/pTα, this pre-TCRcomplex exists together with CD3 on the cell surface. TCRα (or pTα) onthe cell surface has two basic residues in its transmembrane domain, oneof which recruits a CD3γε heterodimer, and another recruits ζζ via theirrespective acidic residues. TCRβ has an additional basic residue in itstransmembrane domain that is believed to recruit CD3δε heterodimer. See,e.g., Kuhns et al. (2006) Deconstructing the Form and Function of theTCR/CD3 Complex, Immunity 24:133-39; Wucherpfennig et al. (2009)Structural Biology of the T-cell Receptor: Insights into ReceptorAssembly, Ligand Recognition, and Initiation of Signaling, Cold SpringHarb. Perspect. Biol. 2:a005140. The assembled complex, comprising TCRαβheterodimer, CD3γε, CD3δε, and ζζ, is expressed on the T cell surface.The polar residues in the transmembrane domain have been suggested toserve as quality control for exiting endoplasmic reticulum; it has beendemonstrated that in the absence of CD3 subunits, TCR chains areretained in the ER and targeted for degradation. See, e.g., Call andWucherpfennig (2005) The T Cell Receptor: Critical Role of the MembraneEnvironment in Receptor Assembly and Function, Annu. Rev. Immunol.23:101-25.

CD3 and ζ chains of the assembled complex provide components for TCRsignaling as TCRαβ heterodimer (or TCRγδ heterodimer) by itself lackssignal transducing activity. The CD3 chains possess oneImmune-Receptor-Tyrosine-based-Activation-Motif (ITAM) each, while thechain contains three tandem ITAMs. ITAMs contain tyrosine residuescapable of being phosphorylated by associated kinases. Thus, theassembled TCR-CD3 complex contains 10 ITAM motifs. See, e.g., Love andHayes (2010) ITAM-Mediated Signaling by the T-Cell Antigen Receptor,Cold Spring Harb. Perspect. Biol. 2:e002485. Following TCR engagement,ITAM motifs are phosphorylated by Src family tyrosine kinases, Lck andFyn, which initiates a signaling cascade, resulting in Ras activation,calcium mobilization, actin cytoskeleton rearrangements, and activationof transcription factors, all ultimately leading to T celldifferentiation, proliferation, and effector actions. Id., see also,Janeway's Immunobiology, 7^(th) Ed., Murphy et al. eds., GarlandScience, 2008; both incorporated herein by reference.

Additionally, TCRβ transmembrane and cytoplasmic domains are thought tohave a role in mitochondrial targeting and induction of apoptosis; infact, naturally occurring N-terminally truncated TCRβ molecules exist inthymocytes. Shani et al. (2009) Incomplete T-cell receptor-β peptidestarget the mitochondrion and induce apoptosis, Blood 113:3530-41. Thus,several important functions are served by the TCR constant region(which, in various embodiments, comprises a portion of extracellular aswell as transmembrane and cytoplasmic domains); and in variousembodiments the structure of this region should be taken intoconsideration when designing humanized TCRs or genetically modifiednon-human animals expressing the same.

Mice transgenic for rearranged T cell receptor sequences are known inthe art. The present invention relates to genetically modified non-humananimals (e.g., rodents, e.g., rats, mice) that comprise unrearrangedhuman or humanized T cell variable gene loci that are capable ofrearranging to form nucleic acid sequences that encode human T cellreceptor variable domains, including animals that comprise T cells thatcomprise rearranged human variable domains and non-human (e.g., mouse orrat) constant regions. The present invention also provides non-humananimals (e.g., rodents, e.g., rats, mice) that are capable of generatinga diverse repertoire of human T cell receptor variable region sequences;thus, the present invention provides non-human animals that express TCRswith fully human variable domains in response to an antigen of interestand that bind an epitope of the antigen of interest. In someembodiments, provided are non-human animals that generate a diverse Tcell receptor repertoire capable of reacting with various antigens,including but not limited to antigens presented by APCs.

In one embodiment, the invention provides genetically modified non-humananimals (e.g., rodents, e.g., rats, mice) that comprise in their genomeunrearranged human TCR variable region segments (V(D)J segments),wherein the unrearranged human TCR variable region segments replace, atan endogenous non-human (e.g., rodent) TCR variable gene locus (e.g.,TCRα, β, δ, and/or γ variable gene locus), endogenous non-human TCRvariable region segments. In one embodiment, unrearranged human TCRvariable gene locus replaces endogenous non-human TCR variable genelocus.

In another embodiment, the invention provides genetically modifiednon-human animals (e.g., rodents, e.g., rats, mice) that comprise intheir genome unrearranged human TCR variable region segments (V(D)Jsegments), wherein the unrearranged human TCR variable region segmentsare operably linked to a non-human TCR constant region gene sequenceresulting in a humanized TCR locus, wherein the humanized TCR locus isat a site in the genome other than the endogenous non-human TCR locus.Thus, in one embodiment, a non-human animal (e.g., rodent, e.g., mouse,rat) comprising a transgene that comprises unrearranged human TCRvariable region segments operably linked to non-human TCR constantregion sequence is also provided.

In one aspect, the genetically modified non-human animals of theinvention comprise in their genome human TCR variable region segments,while retaining non-human (e.g., rodent, e.g., mouse, rat) TCR constantgene segments. In various embodiments, constant regions includetransmembrane domain and the cytoplasmic tail of the TCR. Thus, invarious embodiments of the present invention, the genetically modifiednon-human animals retain endogenous non-human TCR transmembrane domainand cytoplasmic tail. In other embodiments, non-human animals comprisenon-human non-endogenous TCR constant gene sequences, e.g., non-humannon-endogenous TCR transmembrane domain and cytoplasmic tail. Asindicated above, the constant region of the TCR participates in asignaling cascade initiated during antigen-primed T cell activation;thus, endogenous TCR constant region interacts with a variety ofnon-human anchor and signaling proteins in the T cell. Thus, in oneaspect, the genetically modified non-human animals of the inventionexpress humanized T cell receptors that retain the ability to recruit avariety of endogenous non-human anchor or signaling molecules, e.g., CD3molecules (e.g., CD3γ, CD3δ, CD3ε), the ζ chain, Lck, Fyn, ZAP-70, etc.A nonlimiting list of molecules that are recruited to the TCR complex isdescribed in Janeway's Immunobiology, supra. In addition, similar toVELOCIMMUNE® mice, which exhibit normal B cell development and normalclonal selection processes believed to be due at least in part to theplacement of variable regions at the endogenous mouse loci and themaintenance of mouse constant domains, in one aspect, the non-humananimals of the present invention exhibit normal T cell development and Tcell differentiation processes.

In some embodiments, a non-human animal is provided that comprises inits genome unrearranged human TCRα variable region segments, wherein theunrearranged human TCRα variable region segments are operably linked toa non-human TCRα constant region gene sequence resulting in a humanizedTCRα locus. In one embodiment, the humanized TCRα locus is at a site inthe genome other than the endogenous non-human TCRα locus. In anotherembodiment, the unrearranged human TCRα variable region segments replaceendogenous non-human TCRα variable region segments while retainingendogenous non-human TCRα constant region. In one embodiment, theunrearranged human TCRα variable gene locus replaces endogenousnon-human TCRα variable gene locus. In some embodiments, the animalretains endogenous non-human TCRβ variable region and constant regiongene sequences. Thus, the animal expresses a TCR that comprises achimeric human/non-human (i.e., humanized) TCRα chain and a non-humanTCRβ chain.

In other embodiments, a non-human animal is provided that comprises inits genome unrearranged human TCRβ variable region segments, wherein theunrearranged human TCR variable region segments are operably linked to anon-human TCRβ constant region gene sequence resulting in a humanizedTCR locus. In one embodiment, the humanized TCRβ locus is at a site inthe genome other than the endogenous non-human TCRβ locus. In anotherembodiment, the unrearranged human TCRβ variable region segments replaceendogenous non-human TCRβ variable region segments while retainingendogenous non-human TCRβ constant region. In one embodiment, theunrearranged human TCRβ variable gene locus replaces endogenousnon-human TCRβ variable gene locus. In some embodiments, the animalretains endogenous non-human TCRα variable region and constant regiongene sequences. Thus, the animal expresses a TCR that comprises achimeric human/non-human (i.e., humanized) TCRβ chain and a non-humanTCRα chain.

In some specific embodiments, the invention provides a geneticallymodified non-human animal (e.g., rodent, e.g., mouse or rat) thatcomprises in its genome (a) an unrearranged T cell receptor (TCR) avariable gene locus comprising at least one human Vα segment and atleast one human Jα segment, operably linked to an endogenous non-human(e.g., rodent, e.g., mouse or rat) TCRα constant gene sequences, and/or(b) an unrearranged TCRβ variable gene locus comprising at least onehuman Vβ segment, at least one human Dβ segment, and at least one humanJβ segment, operably linked to an endogenous non-human (e.g., rodent,e.g., mouse or rat) TCRβ constant gene sequence.

In various embodiments of the invention, the unrearranged human orhumanized TCR variable gene locus (e.g., TCRα and/or TCRβ variable genelocus gene locus) is comprised in the germline of the non-human animal(e.g., rodent, e.g., mouse or rat). In various embodiments, thereplacements of TCR V(D)J segments by unrearranged human TCR V(D)Jsegments (e.g., Vα and Jα, and/or Vβ and Dβ and Jβ segments) are at anendogenous non-human TCR variable locus (or loci), wherein theunrearranged human V and J and/or V and D and J segments are operablylinked to non-human TCR constant region genes.

In some embodiments of the invention, the non-human animal comprises twocopies of the unrearranged human or humanized TCRα variable gene locusand/or two copies of the unrearranged human or humanized TCRβ variablegene locus. Thus, the non-human animal is homozygous for one or bothunrearranged human or humanized TCRα and TCRβ variable gene locus. Insome embodiments of the invention, the non-human animal comprises onecopy of the unrearranged human or humanized TCRα variable gene locusand/or one copy of the unrearranged human or humanized TCRβ variablegene locus. Thus, the non-human animal is heterozygous for one or bothunrearranged human or humanized TCRα and TCRβ variable gene locus.

In one embodiment, the unrearranged TCRα variable gene locus comprisinghuman variable region segments (e.g., human Vα and Jα segments) ispositioned in the non-human genome such that the human variable regionsegments replace corresponding non-human variable region segments. Inone embodiment, the unrearranged TCRα variable gene locus comprisinghuman variable region segments replaces endogenous TCRα variable genelocus. In one aspect, endogenous non-human Vα and Jα segments areincapable of rearranging to form a rearranged Vα/Jα sequence. Thus, inone aspect, the human Vα and Jα segments in the unrearranged TCRαvariable gene locus are capable of rearranging to form a rearrangedhuman Vα/Jα sequence.

Similarly, in one embodiment, the unrearranged TCRβ variable gene locuscomprising human variable region segments (e.g., human Vβ, Dβ, and Jβsegments) is positioned in the non-human genome such that the humanvariable region segments replace corresponding non-human variable regionsegments. In one embodiment, the unrearranged TCRβ variable gene locuscomprising human variable region segments replaces endogenous TCRβvariable gene locus. In one aspect, endogenous non-human Vβ, Dβ, and Jβsegments are incapable of rearranging to form a rearranged Vβ/Dβ/Jβsequence. Thus, in one aspect, the human Vβ, Dβ, and Jβ segments in theunrearranged TCRβ variable gene locus are capable of rearranging to forma rearranged human Vαβ/Dβ/Jβ sequence.

In yet another embodiment, both the unrearranged TCRα and β variablegene loci comprising human variable region segments replace respectiveendogenous TCRα and β variable gene loci. In one aspect, endogenousnon-human Vα and Jα segments are incapable of rearranging to form arearranged Vα/Jα sequence, and endogenous non-human Vβ, Dβ, and Jβsegments are incapable of rearranging to form a rearranged Vβ/Dβ/Jβsequence. Thus, in one aspect, the human Vα and Jα segments in theunrearranged TCRα variable gene locus are capable of rearranging to forma rearranged human Vα/Jα sequence and the human Vβ, Dβ, and Jβ segmentsin the unrearranged TCRβ variable gene locus are capable of rearrangingto form a rearranged human Vαβ/Dβ/Jβ sequence.

In some aspects of the invention, the non-human animal comprising ahumanized TCRα and/or TCRβ gene locus (comprising an unrearranged TCRαand/or TCRβ variable gene locus) retains an endogenous non-human TCRαand/or TCRβ variable gene locus. In one embodiment, the endogenousnon-human TCRα and/or TCRβ variable gene locus is a non-functionallocus. In one embodiment, the non-functional locus is an inactivatedlocus, e.g., an inverted locus (e.g., the coding nucleic acid sequenceof the variable gene locus is in inverted orientation with respect tothe constant region sequence, such that no successful rearrangements arepossible utilizing variable region segments from the inverted locus). Inone embodiment, the humanized TCRα and/or TCRβ variable gene locus ispositioned between the endogenous non-human TCRα and/or TCRβ variablegene locus and the endogenous non-human TCRα and/or TCRβ constant genelocus.

The number, nomenclature, position, as well as other aspects of V and Jand/or V, D, and J segments of the human and mouse TCR loci may beascertained using the IMGT database, available at www.imgt.org. Themouse TCRα variable locus is approximately 1.5 megabases and comprises atotal of 110Vα and 60 Jα segments (FIG. 2). The human TCRα variablelocus is approximately 1 megabase and comprises a total of 54Vα and 61Jαsegments, with 45Vα and 50Jα believed to be functional. Unless statedotherwise, the numbers of human V(D)J segments referred to throughoutthe specification refers to the total number of V(D)J segments. In oneembodiment of the invention, the genetically modified non-human animal(e.g., rodent, e.g., mouse or rat) comprises at least one human Vα andat least one human Jα segment. In one embodiment, the non-human animalcomprises a humanized TCRα locus that comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, or up to 54 human Vαsegments. In some embodiments, the humanized TCRα locus comprises 2, 8,23, 35, 48, or 54 human Vα segments. Thus, in some embodiments, thehumanized TCRα locus in the non-human animal may comprise 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% 99%, or 100% of human Vα; in some embodiments,it may comprise about 2%, about 3%, about 15%, about 65%, about 90%, or100% of human Vα.

In one embodiment, the non-human animal comprises a humanized TCRα locusthat comprises a DNA fragment comprising a contiguous human sequence ofhuman Vα40 to Vα41 (Vα segment is also referred to as “TRAV” or “TCRAV”)and a DNA fragment comprising a contiguous human sequence of 61 human Jαsegments (Jα segment is also referred to as “TRAJ” or “TCRAJ”). In oneembodiment, the non-human animal comprises a humanized TCRα locus thatcomprises a DNA fragment comprising a contiguous human sequence of humanTRAV35 to TRAV41 and a DNA fragment comprising a contiguous humansequence of 61 human TRAJs. In one embodiment, the non-human animalcomprises a humanized TCRα locus that comprises a DNA fragmentcomprising a contiguous human sequence of human TRAV22 to TRAV41 and aDNA fragment comprising a contiguous human sequence of 61 human TRAJs.In one embodiment, the non-human animal comprises a humanized TCRα locusthat comprises a DNA fragment comprising a contiguous human sequence ofhuman TRAV13-2 to TRAV41 and a DNA fragment comprising a contiguoushuman sequence of 61 human TRAJs. In one embodiment, the non-humananimal comprises a humanized TCRα locus that comprises a DNA fragmentcomprising a contiguous human sequence of human TRAV6 to TRAV41 and 61human TRAJs. In one embodiment, the non-human animal comprises ahumanized TCRα locus that comprises a DNA fragment comprising acontiguous human sequence of human TRAV1-1 to TRAV 41 and 61 humanTRAJs. In various embodiments, the DNA fragments comprising contiguoushuman sequences of human TCRα variable region segments also compriserestriction enzyme sites, selection cassettes, endonucleases sites, orother sites inserted to facilitate cloning and selection during thelocus humanization process. In various embodiments, these additionalsites do not interfere with proper functioning (e.g., rearrangement,splicing, etc.) of various genes at the TCRα locus.

In one embodiment, the humanized TCRα locus comprises 61 human Jαsegments, or 100% of human Jα segments. In a particular embodiment,humanized TCRα locus comprises 8 human Vα segments and 61 human Jαsegments; in another particular embodiment, humanized TCRα locuscomprises 23 human Vα segments and 61 human Jα segments. In anotherparticular embodiment, the humanized TCRα locus comprises a completerepertoire of human Vα and Jα segments, i.e., all human variable aregion gene segments encoded by the a locus, or 54 human Vα and 61 humanJα segments. In various embodiments, the non-human animal does notcomprise any endogenous non-human Vα or Jα segments at the TCRα locus.

The mouse TCRβ variable locus is approximately 0.6 megabases andcomprises a total of 33 Vβ, 2 Dβ, and 14 Jβ segments (FIG. 6). The humanTCRβ variable locus is approximately 0.6 megabases and comprises a totalof 67 Vβ, 2 Dβ, and 14 Jβ segments. In one embodiment of the invention,the genetically modified non-human animal (e.g., rodent, e.g., mouse orrat) comprises at least one human Vβ, at least one human Dβ, and atleast one human Jα segment. In one embodiment, the non-human animalcomprises a humanized TCRβ locus that comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 23, 25, 30, 35, 40, 45, 48, 50, 55, 60, or up to human 67Vβ segments. In some embodiments, the humanized TCRβ locus comprises 8,14, 40, 66, or human 67 Vβ segments. Thus, in some embodiments, thehumanized TCRβ locus in the non-human animal may comprise 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% 99%, or 100% of human Vβ; in some embodiments,it may comprise about 20%, about 60%, about 15%, about 98%, or 100% ofhuman Vβ.

In one embodiment, the non-human animal comprises a humanized TCRβ locusthat comprises a DNA fragment comprising a contiguous human sequence ofhuman Vβ18 to Vβ29-1 (Vβ segment is also referred to as “TRBV” or“TCRBV”). In one embodiment, the non-human animal comprises a humanizedTCRβ locus that comprises a DNA fragment comprising a contiguous humansequence of human TRBV18 to TRBV29-1, a separate DNA fragment comprisinga contiguous human sequence of human Dβ1-Jβ1 (i.e., humanDβ1-Jβ1-1-Jβ1-6 segments), and a separate DNA fragment comprising acontiguous human sequence of human Dβ2-Jβ2 (i.e., human Dβ2-Jβ2-1-Jβ2-7segments). In one embodiment, the non-human animal comprises a humanizedTCRβ locus that comprises a DNA fragment comprising a contiguous humansequence of human TRBV6-5 to TRBV29-1, a separate DNA fragmentcomprising a contiguous human sequence of human Dβ1-Jβ1 (i.e., humanDβ1-Jβ1-1-Jβ1-6 segments), and a separate DNA fragment comprising acontiguous human sequence of human Dβ2-Jβ2 (i.e., human Dβ2-Jβ2-1-Jβ2-7segments). In one embodiment, the non-human animal comprises a humanizedTCRβ locus that comprises a DNA fragment comprising a contiguous humansequence of human TRBV1 to TRBV29-1, a separate DNA fragment comprisinga contiguous human sequence of human Dβ1-Jβ1, and a separate DNAfragment comprising a contiguous human sequence of human Dβ2-Jβ2. In oneembodiment, the non-human animal comprises a humanized TCRβ locus thatcomprises a DNA fragment comprising a contiguous human sequence of humanTRBV1 to TRBV29-1, a separate DNA fragment comprising a contiguous humansequence of human Dβ1-Jβ1, a separate DNA fragment comprising acontiguous human sequence of human Dβ2-Jβ2, and a separate DNA fragmentcomprising the sequence of human TRBV30. In various embodiments, the DNAfragments comprising contiguous human sequences of human TCRβ variableregion segments also comprise restriction enzyme sites, selectioncassettes, endonucleases sites, or other sites inserted to facilitatecloning and selection during the locus humanization process. In variousembodiments, these additional sites do not interfere with properfunctioning (e.g., rearrangement, splicing, etc.) of various genes atthe TCRβ locus.

In one embodiment, the humanized TCRβ locus comprises 14 human Jβsegments, or 100% of human Jβ segments, and 2 human Dβ segments or 100%of human Jβ segments. In another embodiment, the humanized TCRβ locuscomprises at least one human Vβ segment, e.g., 14 human Vβ segments, andall mouse Dβ and Jβ segments. In a particular embodiment, humanized TCRβlocus comprises 14 human Vβ segments, 2 human Dβ segments, and 14 humanJβ segments. In another particular embodiment, the humanized TCRβ locuscomprises a complete repertoire of human Vβ, Dβ, and Jβ segments, i.e.,all human variable β region gene segments encoded by the β locus or 67human Vβ, 2 human Dβ, and 14 human Jβ segments. In one embodiment, thenon-human animal comprises one (e.g., 5′) non-human Vβ segment at thehumanized TCRβ locus. In various embodiments, the non-human animal doesnot comprise any endogenous non-human Vβ, Dβ, or Jβ segments at the TCRβlocus.

In various embodiments, wherein the non-human animal (e.g., rodent)comprises a repertoire of human TCRα and TCRβ (and optionally human TCRδand TCRγ) variable region segments (e.g., a complete repertoire ofvariable region segments), the repertoire of various segments (e.g., thecomplete repertoire of various segments) is utilized by the animal togenerate a diverse repertoire of TCR molecules to various antigens.

In various aspects, the non-human animals comprise contiguous portionsof the human genomic TCR variable loci that comprise V, D, and J, or Dand J, or V and J, or V segments arranged as in an unrearranged humangenomic variable locus, e.g., comprising promoter sequences, leadersequences, intergenic sequences, regulatory sequences, etc., arranged asin a human genomic TCR variable locus. In other aspects, the varioussegments are arranged as in an unrearranged non-human genomic TCRvariable locus. In various embodiments of the humanized TCRα and/or βlocus, the humanized locus can comprise two or more human genomicsegments that do not appear in a human genome juxtaposed, e.g., afragment of V segments of the human V locus located in a human genomeproximal to the constant region, juxtaposed with a fragment of Vsegments of the human V locus located in a human genome at the upstreamend of the human V locus.

In both mouse and human, the TCRδ gene segments are located with theTCRα locus (see FIGS. 2 and 5). TCRδ J and D segments are locatedbetween Vα and Jα segments, while TCRδ V segments are interspersedthroughout the TCRα locus, with the majority located among various Vαsegments. The number and locations of various TCRδ segments can bedetermined from the IMGT database. Due to the genomic arrangement ofTCRδ gene segments within the TCRα locus, successful rearrangement atthe TCRα locus generally deletes the TCRδ gene segments.

In some embodiments of the invention, a non-human animal comprising anunrearranged human TCRα variable gene locus also comprises at least onehuman Vδ segment, e.g., up to complete repertoire of human Vδ segments.Thus, in some embodiments, the replacement of endogenous TCRα variablegene locus results in a replacement of at least one non-human Vδ segmentwith a human Vδ segment. In other embodiments, the non-human animal ofthe invention comprises a complete repertoire of human Vδ, Dδ, and Jδsegments at the unrearranged humanized TCRα locus; in yet otherembodiments, the non-human animal comprises a complete unrearrangedhuman TCRδ locus at the unrearranged humanized TCRα locus (i.e., a TCRδlocus including human variable region segments, as well as humanenhancer and constant region). An exemplary embodiment for constructingan unrearranged humanized TCRα locus comprising complete unrearrangedTCRδ locus is depicted in FIG. 5.

In yet another embodiment, the non-human animal of the invention furthercomprises an unrearranged humanized TCRγ locus, e.g., a TCRγ locuscomprising at least one human Vγ and at least one human Jγ segments(e.g., a complete repertoire of human Vγ and human Jγ variable regionsegments). The human TCRγ locus is on human chromosome 7, while themouse TCRγ locus is on mouse chromosome 13. See the IMGT database formore detail on the TCRγ locus.

In one aspect, the non-human animal (e.g., rodent, e.g., mouse or rat)comprising humanized TCRα and β variable gene loci (and, optionallyhumanized TCRδ/γ variable gene loci) described herein expresses ahumanized T cell receptor comprising a human variable region and anon-human (e.g., rodent, e.g., mouse or rat) constant region on asurface of a T cell. In some aspects, the non-human animal is capable orexpressing a diverse repertoire of humanized T cell receptors thatrecognize a variety of presented antigens.

In various embodiments of the invention, the humanized T cell receptorpolypeptides described herein comprise human leader sequences. Inalternative embodiments, the humanized TCR receptor nucleic acidsequences are engineered such that the humanized TCR polypeptidescomprise non-human leader sequences.

The humanized TCR polypeptides described herein may be expressed undercontrol of endogenous non-human regulatory elements (e.g., rodentregulatory elements), e.g., promoter, silencer, enhancer, etc. Thehumanized TCR polypeptides described herein may alternatively beexpressed under control of human regulatory elements. In variousembodiments, the non-human animals described herein further comprise allregulatory and other sequences normally found in situ in the humangenome.

In various embodiments, the human variable region of the humanized TCRprotein is capable of interacting with various proteins on the surfaceof the same cell or another cell. In one embodiment, the human variableregion of the humanized TCR interacts with MHC proteins (e.g., MHC classI or II proteins) presenting antigens on the surface of the second cell,e.g., an antigen presenting cell (APC). In some embodiments, the MHC Ior II protein is a non-human (e.g., rodent, e.g., mouse or rat) protein.In other embodiments, the MHC I or II protein is a human protein. In oneaspect, the second cell, e.g., the APC, is an endogenous non-human cellexpressing a human or humanized MHC molecule. In a different embodiment,the second cell is a human cell expressing a human MHC molecule.

In one aspect, the non-human animal expresses a humanized T cellreceptor with a non-human constant region on the surface of a T cell,wherein the receptor is capable of interacting with non-human molecules,e.g., anchor or signaling molecules expressed in the T cell (e.g., CD3molecules, the chain, or other proteins anchored to the TCR through theCD3 molecules or the chain).

Thus, in one aspect, a cellular complex is provided, comprising anon-human T-cell that expresses a TCR that comprises a humanized TCRαchain as described herein and humanized TCRβ chain as described herein,and a non-human antigen-presenting cell comprising an antigen bound toan MHC I or MHC II. In one embodiment, the non-human constant TCRα andTCRβ chains are complexed with a non-human zeta (ζ) chain homodimer andCD3 heterodimers. In one embodiment, the cellular complex is an in vivocellular complex. In one embodiment, the cellular complex is an in vitrocellular complex.

The genetically modified non-human animal may be selected from a groupconsisting of a mouse, rat, rabbit, pig, bovine (e.g., cow, bull,buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g.,marmoset, rhesus monkey). For the non-human animals where suitablegenetically modifiable ES cells are not readily available, other methodsare employed to make a non-human animal comprising the geneticmodification. Such methods include, e.g., modifying a non-ES cell genome(e.g., a fibroblast or an induced pluripotent cell) and employingnuclear transfer to transfer the modified genome to a suitable cell,e.g., an oocyte, and gestating the modified cell (e.g., the modifiedoocyte) in a non-human animal under suitable conditions to form anembryo.

In one aspect, the non-human animal is a mammal. In one aspect, thenon-human animal is a small mammal, e.g., of the superfamily Dipodoideaor Muroidea. In one embodiment, the genetically modified animal is arodent. In one embodiment, the rodent is selected from a mouse, a rat,and a hamster. In one embodiment, the rodent is selected from thesuperfamily Muroidea. In one embodiment, the genetically modified animalis from a family selected from Calomyscidae (e.g., mouse-like hamsters),Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae(true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae(climbing mice, rock mice, with-tailed rats, Malagasy rats and mice),Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., molerates, bamboo rats, and zokors). In a specific embodiment, thegenetically modified rodent is selected from a true mouse or rat (familyMuridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment,the genetically modified mouse is from a member of the family Muridae.In one embodiment, the animal is a rodent. In a specific embodiment, therodent is selected from a mouse and a rat. In one embodiment, thenon-human animal is a mouse.

In a specific embodiment, the non-human animal is a rodent that is amouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa,C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, themouse is a 129 strain selected from the group consisting of a strainthat is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm),129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8,129T1, 129T2 (see, e.g., Festing at al. (1999) Revised nomenclature forstrain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al(2000) Establishment and Chimera Analysis of 129/SvEv- andC57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specificembodiment, the genetically modified mouse is a mix of an aforementioned129 strain and an aforementioned C57BL/6 strain. In another specificembodiment, the mouse is a mix of aforementioned 129 strains, or a mixof aforementioned BL/6 strains. In a specific embodiment, the 129 strainof the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, themouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment,the mouse is a mix of a BALB strain and another aforementioned strain.

In one embodiment, the non-human animal is a rat. In one embodiment, therat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment,the rat strain is a mix of two or more strains selected from the groupconsisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and DarkAgouti.

Thus, in one embodiment, the invention provides a genetically modifiedmouse comprising in its genome an unrearranged human or humanized TCRvariable gene locus, e.g., TCRα, TCRβ, TCRδ, and/or TCRγ variable genelocus. In some embodiments, the unrearranged human or humanized TCRvariable gene locus replaces endogenous mouse TCR variable gene locus.In other embodiments, unrearranged human or humanized TCR variable genelocus is at a site in the genome other than the corresponding endogenousmouse TCR locus. In some embodiments, human or humanized unrearrangedTCR variable gene locus is operably linked to mouse TCR constant region.

In one embodiment, a genetically modified mouse is provided, wherein themouse comprises in its genome an unrearranged T cell receptor (TCR) avariable gene locus comprising at least one human Jα segment and atleast one human Vα segment, operably linked to a mouse TCRα constantgene sequence, and an unrearranged TCRβ variable gene locus comprisingat least one human Jβ segment, at least one human Dβ segment, and atleast one human Vβ segment, operably linked to a mouse TCRβ constantgene sequence. In one specific embodiment, the mouse comprises in itsgenome an unrearranged TCRα variable gene locus comprising a completerepertoire of human Jα segments and a complete repertoire of human Vαsegments, operably linked to a mouse TCRα constant gene sequence, and anunrearranged TCRβ variable gene locus comprising a complete repertoireof human Jβ segments, a complete repertoire of human Dβ segments, and acomplete repertoire of human Vβ segments, operably linked to a mouseTCRβ constant gene sequence.

In some embodiments, the unrearranged TCRα variable gene locuscomprising human TCRα variable region segments replaces endogenous mouseTCRα variable gene locus, and the unrearranged TCRβ variable gene locuscomprising human TCRβ variable region segments replaces the endogenousmouse TCRβ variable gene locus. In some embodiments, the endogenousmouse Vα and Jα segments are incapable of rearranging to form arearranged Vα/Jα sequence, and the endogenous mouse Vβ, Dβ, and Jβsegments are incapable of rearranging to form a rearranged Vβ/Dβ/Jβsequence. In some embodiments, the human Vα and Jα segments rearrange toform a rearranged human Vα/Jα sequence, and the human Vβ, Dβ, and Jβsegments rearrange to form a rearranged human Vβ/Dβ/Jβ sequence.

In various embodiments, the non-human animals (e.g., rodents, e.g., miceor rats) described herein produce T cells that are capable of undergoingthymic development, progressing from DN1 to DN2 to DN3 to DN4 to DP andto CD4 or CD8 SP T cells. Such T cells of the non-human animal of theinvention express cell surface molecules typically produced by a T cellduring a particular stage of thymic development (e.g., CD25, CD44, Kit,CD3, pTα, etc.). Thus, the non-human animals described herein expresspTα complexed with TCRβ at the DN3 stage of thymic development. Thenon-human animals described herein express T cells capable of undergoingthymic development to produce CD4+ and CD8+ T cells. Normally, in thethymus the physiological ratio of CD4+ to CD8+ T cells is between about2:1 and 3:1. See, e.g., Ge and Stanley (2008) The O-fucose glycan in theligand-binding domain of Notch 1 regulates embryogenesis and T celldevelopment, Proc. Natl. Acad. Sci. USA 105:1539-44. Thus, in oneembodiment, the non-human animals described herein produce CD4+ and CD8+T cells in the thymus at a ratio of between about 2:1 and 3:1(CD4+:CD8+).

In various embodiments, the non-human animals described herein produce Tcells that are capable of undergoing normal T cell differentiation inthe periphery. In some embodiments, the non-human animals describedherein are capable of producing a normal repertoire of effector T cells,e.g., CTL (cytotoxic T lymphocytes), T_(H)1, T_(H)2, T_(REG), T_(H)17,etc. Thus, in these embodiments, the non-human animals described hereingenerate effector T cells that fulfill different functions typical ofthe particular T cell type, e.g., recognize, bind, and respond toforeign antigens. In various embodiments, the non-human animalsdescribed herein produce effector T cells that kill cells displayingpeptide fragments of cytosolic pathogens expressed in the context of MHCI molecules; recognize peptides derived from antigens degraded inintracellular vesicles and presented by MHC II molecules on the surfaceof macrophages and induce macrophages to kill microorganisms; producecytokines that drive B cell differentiation; activate B cells to produceopsonizing antibodies; induce epithelial cells to produce chemokinesthat recruit neutrophils to infection sites; etc.

In additional embodiments, the non-human animals described hereincomprise a normal number of CD3+ T cells in the periphery, e.g., in thespleen. In some embodiments, the percent of peripheral CD3+ T cells inthe non-human animals described herein is the comparable to that of thewild type animals (i.e., animals comprising all endogenous TCR variableregion segments). In one embodiment, the non-human animals describedherein comprise a normal ratio of splenic CD3+ T cells to totalsplenocytes.

In other aspects, the non-human animals described herein are capable ofgenerating a population of memory T cells in response an antigen ofinterest. For example, the non-human animals generate both centralmemory T cells (Tcm) and effector memory T cells (Tern) to an antigen,e.g., antigen of interest (e.g., antigen being tested for vaccinedevelopment, etc.).

DN1 and DN2 cells that do not receive sufficient signals (e.g., Notchsignals) may develop into B cells, myeloid cells (e.g., dendriticcells), mast cells and NK cells. See, e.g., Yashiro-Ohtani et al. (2010)Notch regulation of early thymocyte development, Seminars in Immunology22:261-69. In some embodiments, the non-human animals described hereindevelop normal numbers of B cells, myeloid cells (e.g., dendriticcells), mast cells and NK cells. In some embodiments, the non-humananimals described herein develop normal dendritic cell population in thethymus.

The predominant type of T cell receptors expressed on the surface of Tcells is TCRα/β, with the minority of the cells expressing TCRδ/γ. Insome embodiments of the invention, the T cells of the non-human animalscomprising humanized TCRα and/or β loci exhibit normal utilization ofTCRα/β and TCRδ/γ loci, e.g., utilization of TCRα/β and TCRδ/γ loci thatis similar to the wild type animal (e.g., the T cells of the non-humananimals described herein express TCRα/β and TCRδ/γ proteins incomparable proportions to that expressed by wild type animals). Thus, insome embodiments, the non-human animals comprising humanized TCRα/β andendogenous non-human TCRδ/γ loci exhibit normal utilization of all loci.

In addition to genetically engineered non-human animals describedherein, a non-human embryo (e.g., a rodent embryo, e.g., mouse or a ratembryo) is also provided, wherein the embryo comprises a donor ES cellthat is derived from a non-human animal (e.g., a rodent, e.g., a mouseor a rat) as described herein. In one aspect, the embryo comprises an ESdonor cell that comprises an unrearranged humanized TCR locus, and hostembryo cells.

Also provided is a tissue, wherein the tissue is derived from anon-human animal (e.g., a mouse or a rat) as described herein, andexpresses a humanized TCR polypeptide (e.g., TCRα and/or TCRβ, or TCRδ,and/or TCRδ polypeptide).

In addition, a non-human cell isolated from a non-human animal asdescribed herein is provided. In one embodiment, the cell is an ES cell.In one embodiment, the cell is a T cell. In one embodiment, the T cellis a CD4+ T cell. In another embodiment, the T cell is a CD8+ T cell.

Also provided is a non-human cell comprising a chromosome or fragmentthereof of a non-human animal as described herein. In one embodiment,the non-human cell comprises a nucleus of a non-human animal asdescribed herein. In one embodiment, the non-human cell comprises thechromosome or fragment thereof as the result of a nuclear transfer.

Also provided is a non-human cell that expresses a TCR proteincomprising a human variable region and a non-human constant region. TheTCR protein may comprise TCRα, TCRβ, or a combination thereof. In oneembodiment, the cell is a T cell, e.g., a CD4+ or a CD8+ T cell.

In one aspect, a non-human induced pluripotent cell comprising anunrearranged humanized TCR locus encoding a humanized TCR polypeptide asdescribed herein is provided. In one embodiment, the induced pluripotentcell is derived from a non-human animal as described herein.

In one aspect, a hybridoma or quadroma is provided, derived from a cellof a non-human animal as described herein. In one embodiment, thenon-human animal is a rodent, e.g., a mouse or rat.

Also provided is a method for making a genetically modified non-humananimal (e.g., rodent, e.g., mouse or rat) described herein. The methodfor making a genetically modified non-human animal results in the animalwhose genome comprises a humanized unrearranged TCR locus (e.g., ahumanized unrearranged TCRα, TCRβ, TCRδ, and/or TCRγ locus). In oneembodiment, a method for making a genetically modified non-human animal(e.g., rodent, e.g., mouse or rat) that expresses a T cell receptorcomprising a human variable region and a non-human (e.g., rodent, e.g.,mouse or rat) constant region on a surface of a T cell is provided,wherein the method comprises replacing in a first non-human animal anendogenous non-human TCRα variable gene locus with an unrearrangedhumanized TCRα variable gene locus comprising at least one human Vαsegment and at least one human Jα segment, wherein the humanized TCRαvariable gene locus is operably linked to endogenous TCRα constantregion; replacing in a second non-human animal an endogenous non-humanTCRβ variable gene locus with an unrearranged humanized TCRβ variablegene locus comprising at least one human Vβ segment, one human Dβsegment, and one human Jβ segment, wherein the humanized TCRβ variablegene locus is operably linked to endogenous TCRβ constant region; andbreeding the first and the second non-human animal to obtain a non-humananimal that expresses a T cell receptor comprising a human variableregion and a non-human constant region. In other embodiments, theinvention provides methods of making a genetically modified non-humananimal whose genome comprises a humanized unrearranged TCRα locus, or anon-human animal whose genome comprises a humanized unrearranged TCRβlocus, generated according to the methods described herein. In variousembodiments, the replacements are made at the endogenous loci. In someembodiments, the method utilizes one or more targeting constructs madeusing VELOCIGENE® technology, introducing the construct(s) into EScells, and introducing targeted ES cell clones into a mouse embryo usingVELOCIMOUSE® technology, as described in the Examples. In someembodiments, the ES cells are derived from a mouse that is a mix of 129and C57BL/6 strains. In various embodiments, the method comprisesprogressive humanization strategy, wherein a construct comprisingadditional variable region segments is introduced into ES cells at eachsubsequent step of humanization, ultimately resulting in a mousecomprising a complete repertoire of human variable region segments (see,e.g., FIGS. 3 and 7).

Thus, nucleotide constructs used for generating genetically engineerednon-human animals described herein are also provided. In one aspect, thenucleotide construct comprises: 5′ and 3′ homology arms, a human DNAfragment comprising human TCR variable region gene segment(s), and aselection cassette flanked by recombination sites. In one embodiment,the human DNA fragment is a TCRα gene fragment and it comprises at leastone human TCRα variable region segment. In another embodiment, the humanDNA fragment is a TCRβ fragment and it comprises at least one human TCRβvariable region gene segment. In one aspect, at least one homology armis a non-human homology arm and it is homologous to non-human TCR locus(e.g., non-human TCRα or TCRβ locus).

A selection cassette is a nucleotide sequence inserted into a targetingconstruct to facilitate selection of cells (e.g., ES cells) that haveintegrated the construct of interest. A number of suitable selectioncassettes are known in the art. Commonly, a selection cassette enablespositive selection in the presence of a particular antibiotic (e.g.,Neo, Hyg, Pur, CM, Spec, etc.). In addition, a selection cassette may beflanked by recombination sites, which allow deletion of the selectioncassette upon treatment with recombinase enzymes. Commonly usedrecombination sites are loxP and Frt, recognized by Cre and Flp enzymes,respectively, but others are known in the art.

In one embodiment, the selection cassette is located at the 5′ end thehuman DNA fragment. In another embodiment, the selection cassette islocated at the 3′ end of the human DNA fragment. In another embodiment,the selection cassette is located within the human DNA fragment, e.g.,within the human intron. In another embodiment, the selection cassetteis located at the junction of the human and mouse DNA fragment.

Various exemplary embodiments of the targeting strategy for generatinggenetically engineered non-human animals, the constructs, and thetargeting vectors used for the same are presented in FIGS. 3, 4, 5, 7,and 8.

Upon completion of gene targeting, ES cells or genetically modifiednon-human animals are screened to confirm successful incorporation ofexogenous nucleotide sequence of interest or expression of exogenouspolypeptide (e.g., human TCR variable region segments). Numeroustechniques are known to those skilled in the art, and include (but arenot limited to) Southern blotting, long PCR, quantitative PCT (e.g.,real-time PCR using TAQMAN®), fluorescence in situ hybridization,Northern blotting, flow cytometry, Western analysis,immunocytochemistry, immunohistochemistry, etc. In one example,non-human animals (e.g., mice) bearing the genetic modification ofinterest can be identified by screening for loss of mouse allele and/orgain of human allele using a modification of allele assay described inValenzuela et al. (2003) High-throughput engineering of the mouse genomecoupled with high-resolution expression analysis, Nature Biotech.21(6):652-659. Other assays that identify a specific nucleotide or aminoacid sequence in the genetically modified animals are known to thoseskilled in the art.

The disclosure also provides a method of modifying a TCR variable genelocus (e.g., TCRα, TCRβ, TCRδ, and/or TCRγ gene locus) of a non-humananimal to express a humanized TCR protein described herein. In oneembodiment, the invention provides a method of modifying a TCR variablegene locus to express a humanized TCR protein on a surface of a T cellwherein the method comprises replacing in a non-human animal anendogenous non-human TCR variable gene locus with an unrearrangedhumanized TCR variable gene locus. In one embodiment wherein the TCRvariable gene locus is a TCRα variable gene locus, the unrearrangedhumanized TCR variable gene locus comprises at least one human Vαsegment and at least one human Jα segment. In one embodiment wherein theTCR variable gene locus is a TCRβ variable gene locus, the unrearrangedhumanized TCR variable gene locus comprises at least one human Vβsegment, at least one human Dβ segment, and at least one human Jβsegment. In various aspects, the unrearranged humanized TCR variablegene locus is operably linked to the corresponding endogenous non-humanTCR constant region.

A humanized TCR protein made by a non-human animal (e.g., rodent, e.g.,mouse or rat) as described herein is also provided, wherein thehumanized TCR protein comprises a human variable region and a non-humanconstant region. Thus, the humanized TCR protein comprises humancomplementary determining regions (i.e., human CDR1, 2, and 3) in itsvariable domain and a non-human constant region.

Although the Examples that follow describe a genetically engineerednon-human animal whose genome comprises humanized TCRα and/or humanizedTCRβ variable gene locus, one skilled in the art would understand that asimilar strategy may be used to produce genetically engineered animalswhose genome comprises humanized TCRδ and/or TCRγ variable gene locus. Agenetically engineered non-human animal with humanization of all fourTCR variable gene loci is also provided.

Use of Genetically Modified TCR Animals

In various embodiments, the genetically modified non-human animals ofthe invention make T cells with humanized TCR molecules on theirsurface, and as a result, would recognize peptides presented to them byMHC complexes in a human-like manner. The genetically modified non-humananimals described herein may be used to study the development andfunction of human T cells and the processes of immunological tolerance;to test human vaccine candidates; to generate TCRs with certainspecificities for TCR gene therapy; to generate TCR libraries to diseaseassociated antigens (e.g., tumor associated antigens (TAAs); etc.

There is a growing interest in T cell therapy in the art, as T cells(e.g., cytotoxic T cells) can be directed to attack and lead todestruction of antigen of interest, e.g., viral antigen, bacterialantigen, tumor antigen, etc., or cells that present it. Initial studiesin cancer T cell therapy aimed at isolation of tumor infiltratinglymphocytes (TILs; lymphocyte populations in the tumor mass thatpresumably comprise T cells reactive against tumor antigens) from tumorcell mass, expanding them in vitro using T cell growth factors, andtransferring them back to the patient in a process called adoptive Tcell transfer. See, e.g., Restifo et al. (2012) Adoptive immunotherapyfor cancer: harnessing the T cell response, Nature Reviews 12:269-81;Linnermann et al. (2011) T-Cell Receptor Gene Therapy: CriticalParameters for Clinical Success, J. Invest. Dermatol. 131:1806-16.However, success of these therapies have thus far been limited tomelanoma and renal cell carcinoma; and the TIL adoptive transfer is notspecifically directed to defined tumor associated antigens (TAAs).Linnermann et al., supra.

Attempts have been made to initiate TCR gene therapy where T cells areeither selected or programmed to target an antigen of interest, e.g., aTAA. Current TCR gene therapy relies on identification of sequences ofTCRs that are directed to specific antigens, e.g., tumor associatedantigens. For example, Rosenberg and colleagues have published severalstudies in which they transduced peripheral blood lymphocytes derivedfrom a melanoma patient with genes encoding TCRα and β chains specificfor melanoma-associated antigen MART-1 epitopes, and used resultingexpanded lymphocytes for adoptive T cell therapy. Johnson et al. (2009)Gene therapy with human and mouse T-cell receptors mediates cancerregression and targets normal tissues expressing cognate antigen, Blood114:535-46; Morgan et al. (2006) Cancer Regression in Patients AfterTransfer of Genetically Engineered Lymphocytes, Science 314:126-29. TheMART-1 specific TCRs were isolated from patients that experienced tumorregression following TIL therapy. However, identification of such TCRs,particularly high-avidity TCRs (which are most likely to betherapeutically useful), is complicated by the fact that most tumorantigens are self antigens, and TCRs targeting these antigens are ofteneither deleted or possess suboptimal affinity, due primarily toimmunological tolerance.

In various embodiments, the present invention solves this problem byproviding genetically engineered non-human animals comprising in theirgenome an unrearranged human TCR variable gene locus. The non-humananimal described herein is capable of generating T cells with a diverserepertoire of humanized T cell receptors. Thus, the non-human animalsdescribed herein may be a source of a diverse repertoire of humanized Tcell receptors, e.g., high-avidity humanized T cell receptors for use inadoptive T cell transfer.

Thus, in one embodiment, the present invention provides a method ofgenerating a T cell receptor to a human antigen comprising immunizing anon-human animal (e.g., a rodent, e.g., a mouse or a rat) describedherein with an antigen of interest, allowing the animal to mount animmune response, isolating from the animal an activated T cell withspecificity for the antigen of interest, and determining the nucleicacid sequence of the T cell receptor expressed by the antigen-specific Tcell.

In one embodiment, the invention provides a method of producing a humanT cell receptor specific for an antigen of interest (e.g., adisease-associated antigen) comprising immunizing a non-human animaldescribed herein with the antigen of interest; allowing the animal tomount an immune response; isolating from the animal a T cell reactive tothe antigen of interest; determining a nucleic acid sequence of a humanTCR variable region expressed by the T cell; cloning the human TCRvariable region into a nucleotide construct comprising a nucleic acidsequence of a human TCR constant region such that the human TCR variableregion is operably linked to the human TCR constant region; andexpressing from the construct a human T cell receptor specific for theantigen of interest. In one embodiment, the steps of isolating a T cell,determining a nucleic acid sequence of a human TCR variable regionexpressed by the T cell, cloning the human TCR variable region into anucleotide construct comprising a nucleic acid sequence of a human TCRconstant region, and expressing a human T cell receptor are performedusing standard techniques known to those of skill the art.

In one embodiment, the nucleotide sequence encoding a T cell receptorspecific for an antigen of interest is expressed in a cell. In oneembodiment, the cell expressing the TCR is selected from a CHO, COS,293, HeLa, PERC.6™ cell, etc.

The antigen of interest may be any antigen that is known to cause or beassociated with a disease or condition, e.g., a tumor associatedantigen; an antigen of viral, bacterial or other pathogenic origin; etc.Many tumor associated antigens are known in the art. A selection oftumor associated antigens is presented in Cancer Immunity (A Journal ofthe Cancer Research Institute) Peptide Database(archive.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm). In someembodiments of the invention, the antigen of interest is a humanantigen, e.g., a human tumor associated antigen. In some embodiments,the antigen is a cell type-specific intracellular antigen, and a T cellreceptor is used to kill a cell expressing the antigen.

In one embodiment, provided herein is a method of identifying a T cellwith specificity against an antigen of interest, e.g., a tumorassociated antigen, comprising immunizing a non-human animal describedherein with the antigen of interest, allowing the animal to mount animmune response, and isolating from the non-human animal a T cell withspecificity for the antigen.

The present invention provides new methods for adoptive T cell therapy.Thus, provided herein is a method of treating or ameliorating a diseaseor condition (e.g., a cancer) in a subject (e.g., a mammalian subject,e.g., a human subject) comprising immunizing a non-human animaldescribed herein with an antigen associated with the disease orcondition, allowing the animal to mount an immune response, isolatingfrom the animal a population of antigen-specific T cells, and infusingisolated antigen-specific T cells into the subject. In one embodiment,the invention provides a method of treating or ameliorating a disease orcondition in a human subject, comprising immunizing the non-human animaldescribed herein with an antigen of interest (e.g., a disease- orcondition-associated antigen, e.g., a tumor associated antigen),allowing the animal to mount an immune response, isolating from theanimal a population of antigen-specific T cells, determining the nucleicacid sequence of a T cell receptor expressed by the antigen-specific Tcells, cloning the nucleic acid sequence of the T cell receptor into anexpression vector (e.g., a retroviral vector), introducing the vectorinto T cells derived from the subject such that the T cells express theantigen-specific T cell receptor, and infusing the T cells into thesubject. In one embodiment, the T cell receptor nucleic acid sequence isfurther humanized prior to introduction into T cells derived from thesubject, e.g., the sequence encoding the non-human constant region ismodified to further resemble a human TCR constant region (e.g., thenon-human constant region is replaced with a human constant region). Insome embodiments, the disease or condition is cancer. In someembodiments, an antigen-specific T cell population is expanded prior toinfusing into the subject. In some embodiments, the subject's immunecell population is immunodepleted prior to the infusion ofantigen-specific T cells. In some embodiments, the antigen-specific TCRis a high avidity TCR, e.g., a high avidity TCR to a tumor associatedantigen. In some embodiments, the T cell is a cytotoxic T cell. In otherembodiments, the disease or condition is caused by a virus or abacterium.

In another embodiment, a disease or condition is an autoimmune disease.T_(REG) cells are a subpopulation of T cells that maintain tolerance toself-antigens and prevent pathological self-reactivity. Thus, alsoprovided herein are methods of treating autoimmune disease that rely ongeneration of antigen-specific T_(REG) cells in the non-human animal ofthe invention described herein.

Also provided herein is a method of treating or ameliorating a diseaseor condition (e.g., a cancer) in a subject comprising introducing thecells affected by the disease or condition (e.g., cancer cells) from thesubject into a non-human animal, allowing the animal to mount an immuneresponse to the cells, isolating from the animal a population of T cellsreactive to the cells, determining the nucleic acid sequence of a T cellreceptor expressed by the T cells, cloning the T cell receptor sequenceinto a vector, introducing the vector into T cells derived from thesubject, and infusing the subject's T cells harboring the T cellreceptor into the subject.

Also provided herein is the use of a non-human animal as describedherein to make nucleic acid sequences encoding human TCR variabledomains (e.g., TCR α and/or β variable domains). In one embodiment, amethod is provided for making a nucleic acid sequence encoding a humanTCR variable domain, comprising immunizing a non-human animal asdescribed herein with an antigen of interest, allowing the non-humananimal to mount an immune response to the antigen of interest, andobtaining therefrom a nucleic acid sequence encoding a human TCRvariable domain that binds the antigen of interest. In one embodiment,the method further comprises making a nucleic acid sequence encoding ahuman TCR variable domain that is operably linked to a non-human TCRconstant region, comprising isolating a T cell from a non-human animaldescribed herein and obtaining therefrom the nucleic acid sequenceencoding TCR variable domain linked to TCR constant region.

Also provided herein is the use of a non-human animal as describedherein to make a human therapeutic, comprising immunizing the non-humananimal with an antigen of interest (e.g., a tumor associated antigen),allowing the non-human animal to mount an immune response, obtainingfrom the animal T cells reactive to the antigen of interest, obtainingfrom the T cells a nucleic acid sequence(s) encoding a humanized TCRprotein that binds the antigen of interest, and employing the nucleicacid sequence(s) encoding a humanized TCR protein in a humantherapeutic.

Thus, also provided is a method for making a human therapeutic,comprising immunizing a non-human animal as described herein with anantigen of interest, allowing the non-human animal to mount an immuneresponse, obtaining from the animal T cells reactive to the antigen ofinterest, obtaining from the T cells a nucleic acid sequence(s) encodinga humanized T cell receptor that binds the antigen of interest, andemploying the humanized T cell receptor in a human therapeutic.

In one embodiment, the human therapeutic is a T cell (e.g., a human Tcell, e.g., a T cell derived from a human subject) harboring a nucleicacid sequence of interest (e.g., transfected or transduced or otherwiseintroduced with the nucleic acid of interest) such that the T cellexpresses the humanized TCR protein with affinity for an antigen ofinterest. In one aspect, a subject in whom the therapeutic is employedis in need of therapy for a particular disease or condition, and theantigen is associated with the disease or condition. In one aspect, theT cell is a cytotoxic T cell, the antigen is a tumor associated antigen,and the disease or condition is cancer. In one aspect, the T cell isderived from the subject.

In another embodiment, the human therapeutic is a T cell receptor. Inone embodiment, the therapeutic receptor is a soluble T cell receptor.Much effort has been expanded to generate soluble T cell receptors orTCR variable regions for use therapeutic agents. Generation of soluble Tcell receptors depends on obtaining rearranged TCR variable regions. Oneapproach is to design single chain TCRs comprising TCRα and TCRβ, and,similarly to scFv immunoglobulin format, fuse them together via a linker(see, e.g., International Application No. WO 2011/044186). The resultingscTv, if analogous to scFv, would provide a thermally stable and solubleform of TCRα/β binding protein. Alternative approaches includeddesigning a soluble TCR having TCRβ constant domains (see, e.g., Chunget al., (1994) Functional three-domain single-chain T-cell receptors,Proc. Natl. Acad. Sci. USA. 91:12654-58); as well as engineering anon-native disulfide bond into the interface between TCR constantdomains (reviewed in Boulter and Jakobsen (2005) Stable, soluble,high-affinity, engineered T cell receptors: novel antibody-like proteinsfor specific targeting of peptide antigens, Clinical and ExperimentalImmunology 142:454-60; see also, U.S. Pat. No. 7,569,664). Other formatsof soluble T cell receptors have been described. The non-human animalsdescribed herein may be used to determine a sequence of a T cellreceptor that binds with high affinity to an antigen of interest, andsubsequently design a soluble T cell receptor based on the sequence.

A soluble T cell receptor derived from the TCR receptor sequenceexpressed by the non-human animal can be used to block the function of aprotein of interest, e.g., a viral, bacterial, or tumor associatedprotein. Alternatively, a soluble T cell receptor may be fused to amoiety that can kill an infected or cancer cell, e.g., a cytotoxicmolecules (e.g., a chemotherapeutic), toxin, radionuclide, prodrug,antibody, etc. A soluble T cell receptor may also be fused to animmunomodulatory molecule, e.g., a cytokine, chemokine, etc. A soluble Tcell receptor may also be fused to an immune inhibitory molecule, e.g.,a molecule that inhibits a T cell from killing other cells harboring anantigen recognized by the T cell. Such soluble T cell receptors fused toimmune inhibitory molecules can be used, e.g., in blocking autoimmunity.Various exemplary immune inhibitory molecules that may be fused to asoluble T cell receptor are reviewed in Ravetch and Lanier (2000) ImmuneInhibitory Receptors, Science 290:84-89, incorporated herein byreference.

The present invention also provides methods for studying immunologicalresponse in the context of human TCR, including human TCR rearrangement,T cell development, T cell activation, immunological tolerance, etc.

Also provided are methods of testing vaccine candidates. In oneembodiment, provided herein is a method of determining whether a vaccinewill activate an immunological response (e.g., T cell proliferation,cytokine release, etc.), and lead to generation of effector, as well asmemory T cells (e.g., central and effector memory T cells).

EXAMPLES

The invention will be further illustrated by the following nonlimitingexamples. These Examples are set forth to aid in the understanding ofthe invention but are not intended to, and should not be construed to,limit its scope in any way. The Examples do not include detaileddescriptions of conventional methods that would be well known to thoseof ordinary skill in the art (molecular cloning techniques, etc.).Unless indicated otherwise, parts are parts by weight, molecular weightis average molecular weight, temperature is indicated in Celsius, andpressure is at or near atmospheric.

Example 1 Generation of Mice with Humanized TCR Variable Gene Loci

Mice comprising a deletion of endogenous TCR (α or β) variable loci andreplacement of endogenous V and J or V, D, and J segments are made usingVELOCIGENE® genetic engineering technology (see, e.g., U.S. Pat. No.6,586,251 and Valenzuela, D. M., et al. (2003) High-throughputengineering of the mouse genome coupled with high-resolution expressionanalysis. Nat. Biotech. 21(6): 652-659), wherein human sequences derivedfrom BAC libraries using bacterial homologous recombination are used tomake large targeting vectors (LTVECs) comprising genomic fragments ofhuman TCR variable loci flanked by targeting arms to target the LTVECsto endogenous mouse TCR variable loci in mouse ES cells. LTVECs relinearized and electroporated into a mouse ES cell line according toValenzuela et al. ES cells are selected for hygromycin or neomycinresistance, and screened for loss of mouse allele or gain of humanallele.

Targeted ES cell clones are introduced into 8-cell stage (or earlier)mouse embryos by the VELOCIMOUSE® method (Poueymirou, W. T. et al.(2007). F0 generation mice fully derived from gene-targeted embryonicstem cells allowing immediate phenotypic analyses. Nat. Biotech. 25:91-99.). VELOCIMICE® (F0 mice fully derived from the donor ES cell)bearing humanized TCR loci are identified by screening for loss ofendogenous TCR variable allele and gain of human allele using amodification of allele assay (Valenzuela et al.). F0 pups are genotypedand bred to homozygosity. Mice homozygous for humanized TCRα and/or TCRβvariable loci (e.g., comprising a subset of human TCRα and/or TCRβvariable segments) are made and phenotyped as described herein.

All mice were housed and bred in the specific pathogen-free facility atRegeneron Pharmaceuticals. All animal experiments were approved by IACUCand Regeneron Pharmaceuticals.

Example 2 Progressive Humanization of TCRα Variable Locus

1.5 megabases of DNA at mouse TCRα locus corresponding to 110 V and 60 Jmouse segments was replaced with 1 megabase of DNA corresponding to 54Vand 61J segments of human TCRα using a progressive humanization strategysummarized in FIGS. 2 and 3. Junctional nucleic acid sequences ofvarious targeting vectors used for progressive humanization strategy ofTCRα locus are summarized in Table 2, and included in the SequenceListing.

TABLE 2 Junctional Nucleic Acid Sequences for Various TCRα LocusTargeting Vectors MAID SEQ ID NO. NO Description 1626 1 Junctionalnucleic acid sequence between the 3′ end of mouse sequence upstream ofthe TCRα variable locus and the 5′ end of loxP-Ub-Hyg-loxP cassette. 2Junctional nucleic acid sequence between the 3′ end of loxP-Ub- Hyg-loxPcassette and the 5′ end of human TCRVα40-TCRVα41- TCRJα1 insertion,including AsiSI site. 3 Junctional nucleic acid sequence between the 3′end of human TCRVα40-TCRVα41-TCRJα1 insertion and the 5′ end of themouse sequence downstream of the human TCRα variable locus, includingNotI site. 1767 4 Junctional nucleic acid sequence between the 3′ end ofmouse sequence upstream of the TCRα variable locus and the 5′ end ofloxP-Ub-Neo-loxP cassette. 5 Junctional nucleic acid sequence betweenthe 3′ end of loxP-Ub- Neo-loxP cassette and the 5′ end of humanTCRVα35-TCRVα39 insertion, including AsiSI site. 1979 6 Junctionalnucleic acid sequence between the 3′ end of mouse sequence upstream ofthe TCRα variable locus and the 5′ end of frt-Pgk-Hyg-frt cassette. 7Junctional nucleic acid sequence between the 3′ end of frt-Pgk- Hyg-frtcassette and the 5′ end of human TCRVα22-TCRVα34 insertion, includingAsiSI site. 1769 8 Junctional nucleic acid sequence between the 3′ endof mouse sequence upstream of the TCRα variable locus and the 5′ end ofloxP-Ub-Neo-loxP cassette. 9 Junctional nucleic acid sequence betweenthe 3′ end of loxP-Ub- Neo-loxP cassette and the 5′ end of humanTCRVα13-2- TCRVα21 insertion, including AsiSI site. 1770 10 Junctionalnucleic acid sequence between the 3′ end of mouse sequence upstream ofthe TCRα variable locus and the 5′ end of loxP-Ub-Hyg-loxP cassette. 11Junctional nucleic acid sequence between the 3′ end of loxP-Ub- Hyg-loxPcassette and the 5′ end of human TCRVα6-TCRVα8-5 insertion, includingAsiSI site. 1771 12 Junctional nucleic acid sequence between the 3′ endof mouse sequence upstream and the TCRα variable locus to the 5′ end ofloxP-Ub-Neo-loxP cassette. 13 Junctional nucleic acid sequence betweenthe 3′ end of loxP-Ub-Neo- loxP cassette and the 5′ end of humanTCRVα1-1-TCRVα5 insertion, including AsiSI site. Human TCRα variableregion segments are numbered as in IMGT database. At least 100 bp ateach junction (about 50 bp from each end) are included in the SequenceListing.

Specifically, as demonstrated in FIG. 4A, DNA from mouse BAC cloneRP23-6A14 (Invitrogen) was modified by homologous recombination and usedas a targeting vector (MAID 1539) to replace TCRAJ1-TCRAJ28 region ofthe endogenous mouse TCRα locus with a Ub-hygromycin cassette followedby a loxP site. DNA from mouse BAC clone RP23-117i19 (Invitrogen) wasmodified by homologous recombination and used as a targeting vector(MAID 1535) to replace ˜15 kb region surrounding (and including) TCRAV1of the endogenous mouse TCRα and δ locus with a PGK-neomycin cassettefollowed by a loxP site. ES cells bearing a double-targeted chromosome(i.e., a single endogenous mouse TCRα locus targeted with both targetingvectors) were confirmed by karyotyping and screening methods (e.g.,TAQMAN™) known in the art. Modified ES cells were treated with CRErecombinase, thereby mediating the deletion of the region between thetwo loxP sites (i.e., the region consisting of the endogenous mouse TCRαlocus from TCRAV1 to TCRAJ1) and leaving behind only a single loxP site,neomycin cassette and the mouse constant and enhancer regions. Thisstrategy resulted in generation of a deleted mouse TCR α/δ locus (MAID1540).

The first human targeting vector for TCRα had 191,660 bp of human DNAfrom the CTD2216p1 and CTD2285 m07 BAC clones (Invitrogen) thatcontained the first two consecutive human TCRαV gene segments (TRAV40 &41) and 61 TCRαJ (50 functional) gene segments. This BAC was modified byhomologous recombination to contain a Not1 site 403 bp downstream (3′)of the TCRαJ1 gene segment for ligation of a 3′ mouse homology arm and a5′ AsiSI site for ligation of a 5′ mouse homology arm. Two differenthomology arms were used for ligation to this human fragment: the 3′homology arm contained endogenous mouse TCRα sequences from theRP23-6A14 BAC clone and the 5′ homology arm contained endogenous TCRαsequence 5′ of mouse TCRαV from mouse BAC clone RP23-117i19. Thismouse-human chimeric BAC was used as a targeting vector (MAID 1626) formaking an initial insertion of human TCRα gene segments plus an upstreamloxp-ub-hygromycin-loxp cassette at the mouse TCRα loci (FIG. 4B). Thejunctional nucleic acid sequences (SEQ ID NOs: 1-3) for the MAID 1626targeting vector are described in Table 2.

Subsequently, a series of human targeting vectors were made thatutilized the same mouse 5′ arm that contained endogenous TCRα sequence5′ of mouse TCRαV from mouse BAC clone RP23-117i19 with alternatingloxP-neomycin-loxP and loxP-hygromycin-loxP (or frt-hygromycin-frt forMAID 1979) selection cassettes.

To generate a human TCRα mini-locus containing a total 8 human TCRαV (7functional) and 61 human TCRαJ (50 functional) gene segments, DNA fromhuman BAC clone RP11-349 μl (Invitrogen) was modified by homologousrecombination and used as a targeting vector (MAID 1767) (FIG. 4C). Thisadded 104,846 bp of human DNA containing the next 6 (5 functional)consecutive human TCRαV gene segments (TRAV35 to TRAV39) and a 5′loxP-ub-neomycin-loxP cassette. Resulting TCRα locus contained a 5′loxp-ub-neomycin-loxP cassette plus a total of 8 human TCRαV (7functional) and 61 human TCRαJ gene segments operably linked to mouseTCRα constant genes and enhancers. The junctional nucleic acid sequences(SEQ ID NOs: 4 and 5) for the MAID 1767 targeting vector are describedin Table 2.

To generate a human TCRα mini-locus containing total of 23 human TCRαV(17 functional) and 61 human TCRαJ gene segments, DNA from mouse BACclone containing from 5′ to 3′: a unique I-Ceul site, a 20 kb mouse TCRAarm 5′ of the mouse TCRA locus to be used for homologous recombinationinto ES cells, and a loxP-Ub-Hyg-loxP cassette in reverse orientation,was modified by bacterial homologous recombination to contain from 5′ to3′: a unique I-Ceul site, a 20 kb mouse TCRA arm 5′ of the mouse TCRAlocus, an frt-pgk-Hyg-frt cassette, and a unique AsiSI site. DNA fromhuman BAC clone RP11-622o20 (Invitrogen), harboring human TCRαV22-V34was modified by homologous recombination to contain a Spec cassetteflanked by unique I-Ceul and AsiSI sites. Subsequently, the Speccassette in the modified human BAC clone was replaced by the sequencecomprised between the I-Ceul and AsiSI sites in the modified mouse BACclone by standard restriction digestion/ligation techniques. Theresulting targeting vector (MAID 1979; FIG. 4D) added 136,557 bp ofhuman DNA that contained the next 15 (10 functional) consecutive humanTCRαJ gene segments (TRAV22 to TRAV34) and a 5′ frt-pgk-Hyg-frtcassette. Resulting TCRα locus contained a 5′ frt-pgk-Hyg-frt cassetteplus a total of 23 human TCRαV (17 functional) and 61 human TCRαV genesegments operably linked to mouse TCRα constant genes and enhancers. Thejunctional nucleic acid sequences (SEQ ID NOs: 6 and 7) for the MAID1979 targeting vector are described in Table 2.

To generate human TCRα mini-locus containing a total of 35 human TCRαV(28 functional) and 61 human TCRαJ gene segments, DNA from human BACclone CTD2501-k5 (Invitrogen) was modified by homologous recombinationand used as a targeting vector (MAID 1769) (FIG. 4E). This added 124,118bp of human DNA that contained the next 12 (11 functional) consecutivehuman TCRαV gene segments (TRAV13-2 to TRAV21) and a5′loxp-ub-neomycin-loxP cassette. Resulting TCRα locus contained a5′loxp-ub-neomycin-loxP cassette plus a total of 35 human TCRαV (28functional) and 61 human TCRαJ gene segments operably linked to mouseTCRα constant genes and enhancers. The junctional nucleic acid sequences(SEQ ID NOs: 8 and 9) for the MAID 1769 targeting vector are describedin Table 2.

To generate a human TCRα mini-locus containing total of 48 human TCRαV(39 functional) and 61 human TCRαJ gene segments, DNA from human BACclone RP11-92F11 (Invitrogen) was modified by homologous recombinationand used as a targeting vector (MAID 1770) (FIG. 4F). This added 145,505bp of human DNA that contained the next 13 (11 functional) consecutivehuman TCRαJ gene segments (TRAV6 to TRAV8.5) and a5′loxp-ub-hygromycin-loxP cassette. Resulting TCRα locus contains a5′loxp-ub-hygromycin-loxP cassette plus a total of 48 human TCRαV (39functional) and 61 human TCRαJ gene segments operably linked to mouseTCRα constant genes and enhancers. The junctional nucleic acid sequences(SEQ ID NOs: 10 and 11) for the MAID 1770 targeting vector are describedin Table 2.

To generate a human TCRα mini-locus containing total of 54 human TCRαV(45 functional) and 61 human TCRαJ gene segments, DNA from human BACclone RP11-780M2 (Invitrogen) was modified by homologous recombinationand used as a targeting vector (MAID 1771) (FIG. 4G). This added 148,496bp of human DNA that contained the next 6 (6 functional) consecutivehuman TCRαV gene segments (TRAV1-1 to TRAV5) and a 5′loxp-ub-neomycin-loxP cassette. Resulting TCRα locus contains a5′loxp-ub-neomycin-loxP cassette plus a total of 54 human TCRαV (45functional) and 61 human TCRαJ gene segment operably linked to mouseTCRα constant genes and enhancers. The junctional nucleic acid sequences(SEQ ID NOs: 12 and 13) for the MAID 1771 targeting vector are describedin Table 2.

In any of the above steps, the selection cassettes are removed bydeletion with Cre or Flp recombinase. In addition, human TCRδ locus maybe introduced as depicted in FIG. 5.

Example 3 Progressive Humanization of TCRβ Variable Locus

0.6 megabases of DNA at mouse TCRβ locus corresponding to 33 V, 2 D, and14 J mouse segments were replaced with 0.6 megabases of DNAcorresponding to 67 V, 2D, and 14 J segments of human TCRβ using aprogressive humanization strategy summarized in FIGS. 6 and 7.Junctional nucleic acid sequences of various targeting vectors used forprogressive humanization strategy of TCRβ locus are summarized in Table3, and included in the Sequence Listing.

TABLE 3 Junctional Nucleic Acid Sequences for Various TCRβ LocusTargeting Vectors MAID SEQ ID NO. NO Description 1625 14 Junctionalnucleic acid sequence between the 3′ end of mouse sequence upstream ofthe TCRβ variable locus (nearby the upstream mouse trypsinogen genes)and the 5′ end of frt-Ub-Neo-frt cassette. 15 Junctional nucleic acidsequence between the 3′ end of frt-Ub- Neo-frt cassette and the 5′ endof human TCRVβ18-TCRVβ29-1 insertion. 16 Junctional nucleic acidsequence between the 3′ end of human TCRVβ18-TCRVβ29-1 insertion and the5′ end of the mouse sequence downstream of the mouse TCRVβ segments(nearby downstream mouse trypsinogen genes). 1715 17 Junctional nucleicacid sequence between 3′ of the downstream mouse trypsinogen genes andthe 5′ end of human TCRDβ1- TCRJβ1-1-TCRJβ1-6 insertion, including lceulsite. 18 Junctional nucleic acid sequence between the 3′ end of humanTCRDβ1-TCRJβ1-1-TCRJβ1-6 insertion and the 5′ end of loxP- Ub-Hyg-loxPcassette. 19 Junctional nucleic acid sequence between the 3′ end ofloxP-Ub- Hyg-loxP cassette and the 5′ end of mouse sequence nearby themouse Cβ1 gene. 20 Junctional nucleic acid sequence between the 3′ endof the mouse sequence nearby the mouse Cβ1 gene and the 5′ end of humanTCRDβ2-TCRJβ2-1-TCRJβ2-7 insertion, including NotI site. 21 Junctionalnucleic acid sequence between the 3′ end of humanTCRDβ2-TCRJβ2-1-TCRJβ2-7 insertion and the 5′ end of the mouse sequencedownstream of the TCRβ variable locus (nearby the Cβ2 mouse sequence).1791 22 Junctional nucleic acid sequence between the 3′ end of mousesequence upstream of the TCRβ variable locus (nearby the upstream mousetrypsinogen genes) and the 5′ end of frt-Ub-Hyg- frt cassette. 23Junctional nucleic acid sequence between the 3′ end of frt-Ub- Hyg-frtcassette and the 5′ end of human TCRVβ6-5-TCRVβ17 insertion. 1792 24Junctional nucleic acid sequence between the 3′ end of mouse sequenceupstream of the TCRβ variable locus (nearby the upstream mousetrypsinogen genes) and the 5′ end of frt-Ub-Neo- frt cassette. 25Junctional nucleic acid sequence between the 3′ end of frt-Ub- Hyg-frtcassette and the 5′ end of human TCRVβ1-TCRVβ12-2 insertion. 6192 26Junctional nucleic acid sequence between the 3′ end of mouse sequencenearby the mouse Cβ2 gene and the 5′ end of the human TCRBV30 exon 2sequence. 27 Junctional nucleic acid sequence between the 3′ end humanTCRBV30 exon 1 sequence and the 5′ end of mouse sequence downstream ofTCRβ locus. Human TCRβ variable region segments are numbered as in IMGTdatabase. At least 100 bp at each junction (about 50 bp from each end)are included in the Sequence Listing.

Specifically, DNA from mouse BAC clone RP23-153p19 (Invitrogen) wasmodified by homologous recombination and used as a targeting vector(MAID 1544) to replace 17 kb region (including TCRBV30) just upstream ofthe 3′ trypsinogen gene cluster in the endogenous mouse TCRβ locus witha PGK-neo cassette followed by a loxP site (FIG. 8A). DNA from mouse BACclone RP23-461h15 (Invitrogen) was modified by homologous recombinationand used as a targeting vector (MAID 1542) to replace 8355 bp region(including TCRBV2 and TCRBV3) downstream of 5′ trypsinogen gene clusterin the endogenous mouse TCRβ locus with a Ub-hygromycin cassettefollowed by a loxP site. ES cells bearing a double-targeted chromosome(i.e., a single endogenous mouse TCRβ locus targeted with both targetingvectors) were confirmed by karyotyping and screening methods (e.g.,TAQMAN™) known in the art. Modified ES cells were treated with CRErecombinase, mediating the deletion of the region between the 5′ and3′loxP sites (consisting of the endogenous mouse TCRβ locus from TCRBV2to TCRBV30) and leaving behind only a single loxP site, hygromycincassette and the mouse TCRBDs, TCRBJs, constant, and enhancer sequences.One mouse TCRVβ was left upstream of the 5′ cluster of trypsinogengenes, and one mouse TCRBβ was left downstream of the mouse Eβ, as notedin FIG. 8A.

The first human targeting vector for TCRβ had 125,781 bp of human DNAfrom the CTD2559j2 BAC clone (Invitrogen) that contained the first 14consecutive human TCRβV gene segments (TRBV18-TRBV29-1). This BAC wasmodified by homologous recombination to contain a 5′ AsiSI site and a 3′Ascl site for ligation of a 5′ and 3′ mouse homology arms. Two differenthomology arms were used for ligation to this human fragment: one set ofhomology arms contained endogenous TCRβ sequence surrounding thedownstream mouse trypsinogen genes from the RP23-153p19 BAC clone andanother set contained endogenous TCRβ sequence surrounding the upstreammouse trypsinogen genes from mouse BAC clone RP23-461h15. Thismouse-human chimeric BAC was used as a targeting vector (MAID 1625) formaking an initial insertion of human TCRβ gene segments plus an upstreamfrt-ub-neomycin-frt cassette at the mouse TCRβ locus, and resulted in ahuman TCRβ mini-locus containing 14 human (8 functional) TCRβV (FIG.8B). The junctional nucleic acid sequences (SEQ ID NOs: 14-16) for theMAID 1625 targeting vector are described in Table 3.

In order to replace mouse TCRβ D and J segments with human TCRβ D and Jsegments, DNA from mouse BAC clone RP23-302p18 (Invitrogen) and fromhuman BAC clone RP11-701D14 (Invitrogen) was modified by homologousrecombination and used as a targeting vector (MAID 1715) into the EScells that contained the TCRβV mini-locus described above (i.e., MAID1625). This modification replaced ˜18540 bp region (from 100 bpdownstream of the polyA of the 3′ trypsinogen genes to 100 bp downstreamfrom the J segments in the D2 cluster which included mouse TCRBD1-J1,mouse constant 1, and mouse TCRBD2-J2) in the endogenous mouse TCR locuswith ˜25425 bp of sequence containing human TCRBD1-J1, loxPUb-hygromycin-loxP cassette, mouse constant 1, human TCRBD2-J2 (FIG.8C(i)). ES cells bearing a double-targeted chromosome (i.e., a singleendogenous mouse TCRβ locus targeted with both targeting vectors) wereconfirmed by karyotyping and screening methods (e.g., TAQMAN™) known inthe art. Modified ES cells were treated with CRE recombinase therebymediating the deletion the hygromycin cassette leaving behind only asingle loxP site downstream from human J segments in D1J cluster (FIG.8C(ii)). The junctional nucleic acid sequences (SEQ ID NOs: 17-21) forthe MAID 1715 targeting vector are described in Table 3.

Subsequently, a series of human targeting vectors were made thatutilized the same mouse 5′ arm that contained endogenous TCR sequencesurrounding the upstream mouse trypsinogen genes from mouse BAC cloneRP23-461h15 with alternating selection cassette.

To generate a human TCRβ mini-locus containing a total 40 human TCRβV(30 functional) and the human TCRβ D and J segments, DNA from human BACclones RP11-134h14 and RP11-785k24 (Invitrogen) was modified byhomologous recombination and combined into a targeting vector (MAID1791) using standard bacterial homologous recombination, restrictiondigestion/ligation, and other cloning techniques. Introduction of theMAID 1791 targeting vector resulted in addition of 198,172 bp of humanDNA that contained the next 26 (22 functional) consecutive human TCRβVgene segments (TRBV6-5 to TRBV17) and a 5′ frt-ub-hygromycin-frtcassette. Resulting TCRβ locus contained a 5′ frt-ub-hygromycin-frtcassette plus a total of 40 human TCRβV (30 functional) and human TCRβ Dand J gene segments operably linked to mouse TCRβ constant genes andenhancers (FIG. 8D). The junctional nucleic acid sequences (SEQ ID NOs:22 and 23) for the MAID 1791 targeting vector are described in Table 3.

To generate a human TCRβ mini-locus containing a total 66 human TCRβV(47 functional) and the human TCRβ D and J segments, DNA from human BACclone RP11-902B7 (Invitrogen) was modified by homologous recombinationand used as a targeting vector (MAID 1792). This resulted in addition of159,742 bp of human DNA that contained the next 26 (17 functional)consecutive human TCRβV gene segments (TRBV1 to TRBV12-2) and a 5′frt-ub-neomycin-frt cassette. Resulting TCRβ locus contained a 5′frt-ub-neomycin-frt cassette plus a total of 66 human TCRβV (47functional) and human TCRβ D and J gene segments operably linked tomouse TCRβ constant genes and enhancers. (FIG. 8E). The junctionalnucleic acid sequences (SEQ ID NOs: 24 and 25) for the MAID 1792targeting vector are described in Table 3.

In any of the above steps, the selection cassettes are removed bydeletion with Cre or Flp recombinase. For example, as depicted in FIG.7, MAID 1716 corresponds to MAID 1715 with the hygromycin cassettedeletion.

Finally, a human TCRβ mini-locus containing a total 67 human TCRβV (48functional) and the human TCRβ D and J segments was generated. MouseTCRBV31 is located ˜9.4 kb 3′ of TCRBC2 (second TCRB constant regionsequence) and is in the opposite orientation to the other TCRBVsegments. The equivalent human V segment is TCRBV30, which is located ina similar position in the human TCRB locus.

To humanize TCRBV31, the mouse BAC clone containing mouse TCRBV31, wasmodified by bacterial homologous recombination to make LTVEC MAID 6192(FIG. 8F). The entire coding region, beginning at the start codon inexon 1, the intron, the 3′ UTR, and the recombination signal sequences(RSS) of TCRBV31 were replaced with the homologous human TCRBV30sequences. The 5′ UTR was kept as mouse sequence. For selection, aself-deleting cassette (lox2372-Ubiquitinpromoter-Hyg-PGKpolyA-Protamine promoter-Cre-SV40polyA-lox2372) wasinserted in the intron (72 bp 3′ of exon 1,289 bp 5′ of exon 2). Forsimplicity, FIGS. 7 and 8 depict the selection cassette 3′ of thehTCRBV30, while it was engineered to be located in the intron betweenexon 1 and exon 2 of the hTCRBV30 gene. The protamine promoter drivingCre expression is transcribed exclusively in post-meiotic spermatids, sothe cassette is “self-deleted” in the F1 generation of mice.

The junctional nucleic acid sequences (SEQ ID NOs: 26 and 27) for theMAID 6192 targeting vector are described in Table 3. MAID 6192 DNA iselectroporated into MAID1792 ES cells. ES cell clones are selected forhygromycin-resistance and screened for loss of mouse TCRB31 allele andgain of human TCRB30 allele.

Similar engineering strategy is used to optionally delete the remaining5′ mouse TCRβ V segment.

Example 4 Generation of TCRα/TCRβ Mice

At each step of progressive humanization of TCRα and TCRβ loci, micehomozygous for humanized TCRα variable locus may be bred with micehomozygous for humanized TCRβ variable locus to form progeny comprisinghumanized TCRα and TCRβ variable loci. Progeny are bred to homozygositywith respect to humanized TCRα and humanized TCRβ loci.

In one embodiment, mice homozygous for humanized TCRα variable locuscomprising 8 human Vα and 61 human Jα (MAID 1767; “1767 HO”) were bredwith mice homozygous for humanized TCRβ variable locus comprising 14human Vβ, 2 human Dβ, and 14 human Jβ (MAID 1716; “1716 HO”). Progenywere bred to homozygosity with respect to both humanized loci.

Example 5 Splenic T Cell Production in Mice Homozygous for HumanizedTCRα and/or TCRβ Locus

Spleens from wild type (WT) mice; mice with deleted mouse TCRα locus(“MAID1540”, see FIG. 3); mice homozygous for human TCRα locus (“MAID1767”, see FIG. 3); mice with deleted TCRβ V segments with the exceptionof two remaining mouse V segments (“MAID1545”, see FIG. 7); micehomozygous for human TCRβ locus, also comprising the two remaining mouseV segments (“MAID 1716”, see FIG. 7); and mice homozygous for both humanTCRα and TCRβ loci, with TCRβ locus also comprising the two remainingmouse V segments (“MAID 1767 1716”) were perfused with Collagenase D(Roche Bioscience) and erythrocytes were lysed with ACK lysis buffer,followed by washing in RPMI medium.

Splenocytes from a single WT, MAID 1540, 1767, 1545, 1716, and 1716 1767representative animal were evaluated by flow cytometry. Briefly, cellsuspensions were made using standard methods. 1×10⁶ cells were incubatedwith anti-mouse CD16/CD32 (2.4G2, BD) on ice for 10 minutes stained withthe appropriate cocktail of antibodies for 30 minutes on ice. Followingstaining, cells were washed and then fixed in 2% formaldehyde. Dataacquisition was performed on an LSRII/CantoII/LSRFortessa flow cytometerand analyzed with FlowJo.

For staining of splenocytes, anti-mouse FITC-CD3 (17A2, BD) was used. Asdemonstrated in FIG. 9, mice with human TCR segments were able toproduce significant numbers of CD3+ T cells, while mice with TCRα mouselocus deletion did not. Mice with TCRβ locus deletion also produced CD3+T cells, presumably due to utilization of the remaining 3′ mouse Vsegment (see below).

Example 6 Thymic T Cell Development in Mice Homozygous for HumanizedTCRα and/or TCRβ Locus

To determine whether mice homozygous for humanized TCRα and/or TCRβlocus exhibited normal T cell development in the thymus, splenocytesfrom four of each WT, 1767 HO, 1716 HO, and 1716 HO 1767 HO age matchedanimals (7-10 weeks old) were used in flow cytometry to evaluateproduction of T cells at various developmental stages, as well as toevaluate frequency and absolute number of each of DN, DP, CD4 SP, andCD8 SP T cells.

Cell type determinations were made based on the presence of CD4, CD8,CD44, and CD25 cell surface markers as summarized in Table 1.Correlation between cell type designation and expression of cell surfacemarkers in the thymus is as follows: double negative (DN) cells (CD4−CD8−), double positive (DP) cells (CD4+ CD8+), CD4 single positive cells(CD4+ CD8−), CD8 single positive cells (CD4− CD8+), double negative1/DN1 cells (CD4− CD8−, CD25− CD44+), double negative 2/DN2 cells (CD4−CD8−, CD25+ CD44+), double negative 3/DN3 cells (CD4− CD8−, CD25+CD44−), double negative 4/DN4 cells (CD4− CD8−, CD25− CD44−).

Thymocytes were evaluated by flow cytometry. Briefly, cell suspensionswere made using standard methods. Flow cytometry was conducted asdescribed in Example 5. Antibodies used were: anti-mouse PE-CD44 (IM7,BioLegend), PeCy7-CD25 (PC61, BioLegend), APC-H7-CD8a (53-6.7, BD), andAPC-CD4 (GK1.5, eBioscience).

As shown in FIGS. 10 and 11, mice homozygous for humanized TCRα, TCRβ,and both TCRα and TCRβ were able to produce a DN1, DN2, DN3, DN4, DP,CD4 SP, and CD8 SP T cells, indicating that the T cells produced fromthe humanized loci are capable of undergoing T cell development in thethymus.

Example 7 Splenic T Cell Differentiation in Mice Homozygous forHumanized TCRα and/or TCRβ Locus

To determine whether mice homozygous for humanized TCRα and/or TCRβlocus exhibited normal T cell differentiation in the periphery (e.g.,spleen), four of each WT, 1767 HO, 1716 HO, and 1716 HO 1767 HO agematched animals (7-10 weeks old) were used in flow cytometry to evaluateproduction of various T cell types in the spleen (CD3+, CD4+, CD8+, TnaïTcm, and Teff/em), as well as to evaluate the absolute number of eachT cell type in the spleen.

Cell type determinations were made based on the presence of CD19 (B cellmarker), CD3 (T cell marker), CD4, CD8, CD44, and CD62L (L-selectin)cell surface markers. Correlation between cell type designation andexpression of cell surface markers in the spleen is as follows: T cells(CD3+), CD4 T cells (CD3+ CD4+ CD8−), CD8 T cells (CD3+ CD4− CD8+), CD4effector/effector memory T cells (CD3+ CD4+ CD8− CD62L− CD44+), CD4central memory T cells (CD3+ CD4+ CD8− CD62L+ CD44+), CD4 naïve T cells(CD3+ CD4+ CD8− CD62L+ CD44−), CD8 effector/effector memory T cells(CD3+ CD4− CD8+ CD62L− CD44+), CD8 central memory T cells (CD3+ CD4−CD8+ CD62L+ CD44+), CD8 naïve T cells (CD3+ CD4− CD8+ CD62L+ CD44−).

Splenocytes were evaluated by flow cytometry. Briefly, cell suspensionswere made using standard methods. Flow cytometry was conducted asdescribed in Example 5. Antibodies used were: anti-mouse FITC-CD3 (17A2,BD), PE-CD44 (IM7, BioLegend), PerCP-Cy5.5-CD62L (Mel-14, BioLegend),APC-H7-CD8a (53-6.7, BD), APC-CD4 (GK1.5, eBioscience), and V450-CD19(1D3, BD).

As shown in FIGS. 12-14, T cells in the spleen of mice homozygous forhumanized TCRα, TCRβ, and both TCRα and TCRβ were able to undergo T celldifferentiation, and both CD4+ and CD8+ T cells were present. Inaddition, memory T cells were detected in the spleens of the micetested.

Example 8 Utilization of Human V Segments in Humanized TCR Mice

Expression of human TCRβ V segments was evaluated on protein and RNAlevel using flow cytometry and TAQMAN™ real-time PCR, respectively, inmice homozygous for humanized TCRβ locus (1716 HO) and mice homozygousfor both humanized TCRβ and TCRα locus (1716 HO 1767 HO).

For flow cytometry, splenic T cell were prepared and analysis conductedas described in Example 5. For flow cytometry, TCRβ repertoire kit(IOTEST® Beta Mark, Beckman Coulter) was used. The kit containsanti-human antibodies specific for a number of human TCRBVs, e.g.,hTRBV-18, -19, -20, -25, -27, -28, and -29.

Results are summarized in FIG. 15. The tables presented in FIG. 15A (CD8T cell overlay) and FIG. 15B (CD4 T cell overlay) demonstrate thatsplenic T cells in both 1716 HO and 1716 HO 1767 HO mice utilized anumber of human TCRβ V segments. The wild type mice were used as anegative control.

For real-time PCR, total RNA was purified from spleen and thymus usingMAGMAX™-96 for Microarrays Total RNA Isolation Kit (Ambion by LifeTechnologies) according to manufacturer's specifications. Genomic DNAwas removed using MAGMAX™TURBO™DNase Buffer and TURBO DNase from theMAGMAX kit listed above (Ambion by Life Technologies). mRNA (up to 2.5ug) was reverse-transcribed into cDNA using SUPERSCRIPT® VILO™ MasterMix (Invitrogen by Life Technologies). cDNA was diluted to 2-5 ng/μL,and 10-25 ng cDNA was amplified with the TAQMAN® Gene Expression MasterMix (Applied Biosystems by Life Technologies) using the ABI 7900HTSequence Detection System (Applied Biosystems), using the primers andTaqman MGB probes (Applied Biosystems) or BHQ1/BHQ-Plus probes(Biosearch Technologies) depicted in Table 4 according to manufacturer'sinstructions. The relative expression of each gene was normalized to themurine TCR beta constant 1 (TRBC1) control.

TABLE 4  Primers and Probes Used for Detecting RNA Expression of TCRβV Segmentsand Constant Region in Humanized TCR Mice by Real-Time PCR (TAQMAN ™)Sense Primer (5′-3′) Antisense Primer (5′-3′) Probe (5′-3′) SED SED SEDTRBV Sequence ID NO Sequence ID NO Sequence ID NO hTRBV CCGGCGTCATGC 28GGGCTGCATCTCAGT 29 FAM- 30 18 AGAA CTTGC CACCTGGTCAGG AGGAGG-MGB hTRBVGGAATCACTCAG 31 ATTCTGTTCACAACTC 32 FAM- 33 19 TCCCCAAAG AGGGTCATCAGAAAGGAAG GACAGAAT-MGB hTRBV CGAGCAAGGCGT 34 GGACAAGGTCAGGCT 35 FAM-36 20 CGAGAA TGCA ACAAGTTTCTCAT CAACC-MGB hTRBV TGTTACCCAGAC 37TCTGAGAACATTCCA 38 FAM- 39 24 CCCAAGGA GCATAATCCT TAGGATCACAAAGACAGGAA-MGB hTRBV TCCCCTGACCCT 40 TGCTGGCACAGAGGT 41 FAM- 42 25 GGAGTCTACTGAGA CAGGCCCTCACAT AC-MGB hTRBV AAGCCCAAGTGA 43 ATTCTGAGAACAAGT 44FAM- 45 27 CCCAGAA CACTGTTAACTTC CTCATCACAGTGA CTGGAA-MGB hTRBVGTGAAAGTAACC 46 ATCCTGGACACATTC 47 FAM- 48 28 CAGAGCTCGAG CAGAAAAACATATCTAGTCAAA AGGACGGGA-MGB hTRBV TGTCATTGACAA 49 TGCTGTCTTCAGGGC 50FAM- 51 29 GTTTCCCATCAG TCATG TCAACTCTGACTG TGAGCA-MGB mTRBCAGCCGCCTGAGG 52 GCCACTTGTCCTCCT 53 FAM- 54 1 GTCTCT CTGAAAGTACCTTCTGGCAC AATCCTCGCA- BHQ

As demonstrated in FIGS. 16A-B, mice homozygous for humanized TCRβ locus(1716 HO) and mice homozygous for both humanized TCRβ and TCRα locus(1716 HO 1767 HO) exhibited RNA expression of various human TCRβsegments in both the thymus and the spleen. Mice also exhibited RNAexpression of mouse TRBV-1 and TRBV-31 segments (data not shown), but nomouse TRBV-1 protein was detected by flow cytometry (data not shown).

Mouse TRBV-31 segment is replaced with human TRBV-30 segment asdemonstrated in FIG. 8F, and mice are generated from MAID 6192 ES cellsas described herein. The spleens and thymi of resulting homozygousanimals are tested for utilization of human V8 segments, includingTRBV-30, by flow cytometry and/or real-time PCR as described herein.mTRBV-1 segment may also be deleted.

Example 9 T Cell Development in Mice Homozygous for 23 Human TCR VαSegments

Homozygous humanized TCRα mice characterized in the previous examplescontained 8 human Vα segments and 61 human Jα segments (1767 HO, seeFIG. 3). Homozygous humanized TCRα mice comprising 23 human Vα segmentsand 61 human Jα segments (1979 HO, see FIG. 3) were tested for theirability to generate splenic CD3+ T cells and exhibit T cell developmentin the thymus.

Experimental data was obtained using flow cytometry using appropriateantibodies as described in the preceding examples. As depicted in FIG.17, a mouse homozygous for 23 human Vα segments and 61 human Jα segmentsproduced a significant number of splenic CD3+ T cells, and the percentof peripheral CD3+ T cells was comparable to that of the wild typeanimals (FIG. 19).

Moreover, thymocytes in 1979 HO mice were able to undergo T celldevelopment and contained T cells at DN1 DN2, DN3, DN4, DP, CD4 SP, andCD8 SP stages (FIG. 18).

Example 10 T Cell Development and Differentiation in Mice Homozygous fora Complete Repertoire of Both Human TCRα and TCR β Variable RegionSegments

Mice homozygous for a complete repertoire of human TCRα variable regionsegments (i.e., 54 human Vα and 61 human Jα) and homozygous for acomplete repertoire of human TCRβ variable region segments (67 human Vβ,2 human Dβ, and 14 human Jβ), “1771 HO 6192 HO” (see FIGS. 3 and 7), aretested for their ability to produce thymocytes that undergo normal Tcell development, produce T cells that undergo normal T celldifferentiation in the periphery, and utilize the complete repertoire oftheir human Vα and Vβ segments.

Flow cytometry is conducted to determine the presence of DN1, DN2, DN3,DN4, DP, CD4 SP and CD8 SP T cells in the thymus using anti-mouse CD4,CD8, CD25, and CD44 antibodies as described above in Examples 5 and 6.Flow cytometry is also conducted to determine the number of CD3+ T cellsin the periphery, as well as to evaluate T cell differentiation in theperiphery (e.g., presence of effector and memory T cells in theperiphery). The experiment is conducted using anti-mouse CD3, CD19, CD4,CD8, CD44, and CD62L antibodies as described above in Examples 5 and 7.

Finally, flow cytometry and/or real-time PCR are conducted to determinewhether T cells in 1771 HO 6192 HO mice utilize a complete repertoire ofTCRB and TCRA V segments. For protein expression using flow cytometry,TCRβ repertoire kit (IOTEST® Beta Mark, Beckman Coulter), containinganti-human hTCRBV-specific antibodies, is utilized (see Example 8). ForRNA expression using real-time PCR, cDNAs from spleens or thymi areamplified using human TCR-V primers and Taqman probes, according tomanufacturers instructions and as described above in Example 8.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Entire contents of all non-patent documents, patent applications andpatents cited throughout this application are incorporated by referenceherein in their entirety.

What is claimed is:
 1. A genetically modified non-human animal,comprising in its genome: an unrearranged T cell receptor (TCR) avariable gene locus comprising at least one human Vα segment and atleast one human Jα segment, operably linked to a non-human TCRα constantgene sequence.
 2. The animal of claim 1, wherein the unrearranged TCRαvariable gene locus replaces an endogenous non-human TCRα variable genelocus.
 3. The animal of claim 1, wherein endogenous non-human Vα and Jαsegments are incapable of rearranging to form a rearranged Vα/Jαsequence.
 4. The animal of claim 1, wherein the animal lacks afunctional endogenous non-human TCRα variable locus.
 5. The animal ofclaim 4, wherein the lack of the functional endogenous non-human TCRαvariable locus comprises a deletion selected from the group consistingof (a) a deletion of all endogenous Vα gene segments, (b) a deletion ofall endogenous Jα gene segments, and (c) a combination thereof.
 6. Theanimal of claim 1, wherein the human Vα and Jα segments rearrange toform a rearranged human Vα/Jα sequence.
 7. The animal of claim 6,wherein the animal expresses a T cell receptor comprising a human TCRαvariable region on the surface of a T cell.
 8. The animal of claim 1,wherein T cells of the animal undergo thymic T cell development toproduce CD4 and CD8 single positive T cells.
 9. The animal of claim 1,wherein the animal comprises a normal ratio of splenic CD3+ T cells tototal splenocytes.
 10. The animal of claim 1, wherein the animalgenerates a population of central and effector memory T cells to anantigen of interest.
 11. The animal of claim 1, wherein the unrearrangedTCRα variable gene locus comprises a complete repertoire of human Jαsegments and a complete repertoire of human Vα segments.
 12. The animalof claim 1, wherein the animal retains an endogenous non-human TCRαvariable gene locus, and wherein the locus is a non-functional locus.13. The animal of claim 1, wherein the animal is a rodent.
 14. Theanimal of claim 13, wherein the rodent is a mouse.
 15. A geneticallymodified non-human animal, comprising in its genome: an unrearrangedTCRβ variable gene locus comprising at least one human Vβ segment, atleast one human Dβ segment, and at least one human Jβ segment, operablylinked to a non-human TCRβ constant gene sequence.
 16. The animal ofclaim 15, wherein the unrearranged TCRβ variable gene locus replaces anendogenous non-human TCRβ variable gene locus.
 17. The animal of claim15, wherein endogenous rodent Vβ, Dβ, and Jβ segments are incapable ofrearranging to form a rearranged Vβ/Dβ/Jβ sequence.
 18. The animal ofclaim 15, wherein the animal lacks a functional endogenous non-humanTCRβ variable locus.
 19. The animal of claim 18, wherein the lack of thefunctional endogenous rodent TCRβ variable locus comprises a deletionselected from the group consisting of (a) a deletion of all endogenousVβ gene segments, (b) a deletion of all endogenous Dβ gene segments, (c)a deletion of all endogenous Jβ gene segments, and (d) a combinationthereof.
 20. The animal of claim 15, wherein the human Vβ, Dβ, and Jβsegments rearrange to form a rearranged human Vβ/Dβ/Jβ sequence.
 21. Theanimal of claim 20, wherein the animal expresses a T cell receptorcomprising a human TCRβ variable region on the surface of a T cell. 22.The animal of claim 15, wherein T cells of the animal undergo thymic Tcell development to produce CD4 and CD8 single positive T cells.
 23. Theanimal of claim 15, wherein the animal comprises a normal ratio ofsplenic CD3+ T cells to total splenocytes.
 24. The animal of claim 15,wherein the animal generates a population of central and effector memoryT cells to an antigen of interest.
 25. The animal of claim 15, whereinthe unrearranged TCRβ variable gene locus comprises a completerepertoire of human Jβ segments, a complete repertoire of human Dβsegments, and a complete repertoire of human Vβ segments.
 26. The animalof claim 15, wherein the animal retains an endogenous non-human TCRβvariable gene locus, and wherein the locus is a non-functional locus.27. The animal of claim 15, wherein the animal is a rodent.
 28. Theanimal of claim 27, wherein the rodent is a mouse.
 29. A geneticallymodified non-human animal, comprising in its genome: an unrearranged Tcell receptor (TCR) a variable gene locus comprising at least one humanVα segment and at least one human Jα segment, operably linked to anon-human TCRα constant gene sequence; and, an unrearranged TCRβvariable gene locus comprising at least one human Vβ segment, at leastone human Dβ segment, and at least one human Jβ segment, operably linkedto a non-human TCRβ constant gene sequence.
 30. The animal of claim 29,wherein the unrearranged TCRα variable gene locus replaces an endogenousnon-human TCRα variable gene locus, and wherein the unrearranged TCRβvariable gene locus replaces an endogenous non-human TCRβ variable genelocus.
 31. The animal of claim 29, wherein endogenous non-human Vα andJα segments are incapable of rearranging to form a rearranged Vα/Jαsequence, and wherein endogenous non-human Vβ, Dβ, and Jβ segments areincapable of rearranging to form a rearranged Vβ/Dβ/Jβ sequence.
 32. Theanimal of claim 29, wherein the animal lacks a functional endogenousnon-human TCRα variable locus and lacks a functional endogenousnon-human TCRβ variable locus.
 33. The animal of claim 32, wherein thelack of the functional endogenous non-human TCRα variable locuscomprises a deletion selected from the group consisting of (a) adeletion of all endogenous Vα gene segments, (b) a deletion of allendogenous Jα gene segments, and (c) a combination thereof; and the lackof the functional endogenous non-human TCRβ variable locus comprises adeletion selected from the group consisting of (a) a deletion of allendogenous Vβ gene segments, (b) a deletion of all endogenous Dβ genesegments, (c) a deletion of all endogenous Jβ gene segments, and (d) acombination thereof.
 34. The animal of claim 29, wherein the human Vαand Jα segments rearrange to form a rearranged human Vα/Jα sequence, andthe human Vβ, Dβ, and Jβ segments rearrange to form a rearranged humanVβ/Dβ/Jβ sequence.
 35. The animal of claim 34, wherein the animalexpresses a T cell receptor comprising a human variable region and anon-human constant region on a surface of a T cell.
 36. The animal ofclaim 29, wherein T cells of the animal undergo thymic T celldevelopment to produce CD4 and CD8 single positive T cells.
 37. Theanimal of claim 29, wherein the animal comprises a normal ratio ofsplenic CD3+ T cells to total splenocytes.
 38. The animal of claim 29,wherein the animal generates a population of central and effector memoryT cells to an antigen of interest.
 39. The animal of claim 29, whereinthe unrearranged TCRα variable gene locus comprises 61 human Jα segmentsand 8 human Vα segments, and wherein the unrearranged TCRβ variable genelocus comprises 14 human segments, 2 human Dβ segments, and 14 human Vβsegments.
 40. The animal of claim 29, wherein the unrearranged TCRαvariable gene locus comprises a complete repertoire of human Jα segmentsand a complete repertoire of human Vα segments, and wherein theunrearranged TCRβ variable gene locus comprises a complete repertoire ofhuman 4 segments, a complete repertoire of human Dβ segments, and acomplete repertoire of human Vβ segments.
 41. The animal of claim 29,wherein the animal retains endogenous non-human TCRα and TCRβ variablegene loci, and wherein the loci are non-functional loci.
 42. The animalof claim 29, wherein the animal further comprises nucleotide sequencesof human Vδ segments at a humanized TCRα locus.
 43. The animal of claim42, wherein the animal further comprises a complete repertoire of humanVδ segments, a complete repertoire of human Dδ segments, and a completerepertoire of human Jδ segments at the humanized TCRα locus.
 44. Theanimal of claim 29, wherein the animal is a rodent.
 45. The animal ofclaim 44, wherein the rodent is a mouse.
 46. A method for making agenetically modified non-human animal that expresses a T cell receptorcomprising a human variable region and a non-human constant region on asurface of a T cell comprising: replacing in a first non-human animal anendogenous non-human TCRα variable gene locus with an unrearrangedhumanized TCRα variable gene locus comprising at least one human Vαsegment and at least one human Jα segment to generate a humanized TCRαvariable gene locus, wherein the humanized TCRα variable gene locus isoperably linked to endogenous non-human TCRα constant region; replacingin a second non-human animal an endogenous non-human TCRβ variable genelocus with an unrearranged humanized TCRβ variable gene locus comprisingat least one human Vβ segment, at least one human Dβ segment, and atleast one human Jβ segment to generate a humanized TCRβ variable genelocus, wherein the humanized TCRβ variable gene locus is operably linkedto endogenous non-human TCRβ constant region; and breeding the first andthe second animal to obtain a non-human animal that expresses a T cellreceptor comprising a human variable region and a non-human constantregion.
 47. The method of claim 46, wherein endogenous non-human Vα andJα segments are incapable of rearranging to form a rearranged Vα/Jαsequence, and wherein endogenous non-human Vβ, Dβ, and Jβ segments areincapable of rearranging to form a rearranged Vβ/Dβ/Jβ sequence.
 48. Themethod of claim 46, wherein the genetically modified animal lacks afunctional endogenous non-human TCRα variable locus and lacks afunctional endogenous non-human TCRβ variable locus.
 49. The method ofclaim 48, wherein the lack of the functional endogenous non-human TCRαvariable locus comprises a deletion selected from the group consistingof (a) a deletion of all endogenous Vα gene segments, (b) a deletion ofall endogenous Jα gene segments, and (c) a combination thereof; and thelack of the functional endogenous non-human TCRβ variable locuscomprises a deletion selected from the group consisting of (a) adeletion of all endogenous Vβ gene segments, (b) a deletion of allendogenous Dβ gene segments, (c) a deletion of all endogenous Jβ genesegments, and (d) a combination thereof.
 50. The method of claim 46,wherein the human Vα and Jα segments rearrange to form a rearrangedhuman Vα/Jα sequence, and the human Vβ, Dβ, and Jβ segments rearrange toform a rearranged human Vβ/Dβ/Jβ sequence.
 51. The method of claim 46,wherein T cells of the rodent undergo thymic T cell development toproduce CD4 and CD8 single positive T cells.
 52. The method of claim 46,wherein the animal comprises a normal ratio of splenic CD3+ T cells tototal splenocytes.
 53. The method of claim 46, wherein the animalgenerates a population of central and effector memory T cells to anantigen of interest.
 54. The method of claim 46, wherein theunrearranged humanized TCRα variable gene locus comprises 61 human Jαsegments and 8 human Vα segments, and wherein the unrearranged humanizedTCRβ variable gene locus comprises 14 human Jβ segments, 2 human Dβsegments, and 14 human Vβ segments.
 55. The method of claim 46, whereinthe unrearranged humanized TCRα variable gene locus comprises a completerepertoire of human Jα segments and a complete repertoire of human Vαsegments, and wherein the unrearranged humanized TCRβ variable genelocus comprises a complete repertoire of human Jβ segments, a completerepertoire of human Dβ segments, and a complete repertoire of human Vβsegments.
 56. The method of claim 46, wherein the non-human animal is arodent.
 57. The method of claim 56, wherein the rodent is a mouse.
 58. Agenetically modified mouse comprising in its genome: an unrearranged Tcell receptor (TCR) a variable gene locus comprising a completerepertoire of human Jα segments and a complete repertoire of human Vαsegments, operably linked to a mouse TCRα constant gene sequence, and anunrearranged TCRβ variable gene locus comprising a complete repertoireof human Jβ segments, a complete repertoire of human Dβ segments, and acomplete repertoire of human Vβ segments, operably linked to a mouseTCRβ constant gene sequence.
 59. The mouse of claim 58, wherein theunrearranged TCRα variable gene locus replaces endogenous mouse TCRαvariable gene locus, and wherein the unrearranged TCRβ variable genelocus replaces the endogenous mouse TCRβ variable gene locus.
 60. Themouse of claim 58, wherein the endogenous mouse Vα and Jα segments areincapable of rearranging to form a rearranged Vα/Jα sequence, andwherein the endogenous mouse Vβ, Dβ, and Jβ segments are incapable ofrearranging to form a rearranged Vβ/Dβ/Jβ sequence.
 61. The mouse ofclaim 58, wherein the human Vα and Jα segments rearrange to form arearranged human Vα/Jα sequence, and the human Vβ, Dβ, and Jβ segmentsrearrange to form a rearranged human Vβ/Dβ/Jβ sequence.
 62. The mouse ofclaim 61, wherein the mouse expresses a T cell receptor comprising ahuman variable region and a mouse constant region on a surface of a Tcell.
 63. The mouse of claim 58, wherein T cells of the mouse undergothymic T cell development to produce CD4 and CD8 single positive Tcells.
 64. The mouse of claim 58, wherein the mouse comprises a normalratio of splenic CD3+ T cells to total splenocytes.
 65. The mouse ofclaim 58, wherein the mouse generates a population of central andeffector memory T cells to an antigen of interest.
 66. The mouse ofclaim 58, wherein the mouse further comprises a complete repertoire ofVδ, a complete repertoire of Dδ, and a complete repertoire of Jδsegments at a humanized TCRα locus.
 67. The mouse of claim 58, whereinthe mouse retains endogenous mouse TCRα and TCRβ variable gene loci, andwherein the loci are non-functional loci.
 68. A method of producing ahuman T cell receptor to an antigen of interest comprising: immunizing anon-human animal of claim 1 with the antigen of interest; allowing theanimal to mount an immune response; isolating from the animal a T cellreactive to the antigen of interest; determining a nucleic acid sequenceof a human TCR variable region expressed by the T cell; cloning thehuman TCR variable region into a nucleotide construct comprising anucleic acid sequence of a human TCR constant region, wherein the humanTCR variable region is operably linked to the human TCR constant region;and expressing a human T cell receptor in a cell.